(Advances in Protein Chemistry 69) Wei Yang (Eds.) - DNA Repair and Replication-Elsevier, Academic Press (2004) PDF

ADVANCES IN PROTEIN CHEMISTRY
Volume 69
DNA Repair and Replication

This Page Intentionally Left Blank
ADVANCES IN
PROTEIN CHEMISTRY
EDITED BY
FREDERIC M. RICHARDS DAVID S. EISENBERG

Department of Molecular Biophysics Department of Chemistry and Biochemistry
and Biochemistry, Center for Genomics and Proteomics
Yale University University of California, Los Angeles
New Haven, Connecticut Los Angeles, California
JOHN KURIYAN
Department of Molecular and Cellular Biology
University of California, Berkeley
Berkeley, California
VOLUME 69
DNA Repair and Replication

EDITED BY
Wei Yang
Section Chief of Structure and Mechanism,
Laboratory of Molecular Biology, NIDDK, NIH,
Bethesda, Maryland
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CONTENTS
PREFACE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
Base Excision Repair
J. Christopher Fromme and Gregory L. Verdine
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
II. Types of Damage Repaired by BER . . . . . . . . . . . . . . . . . . . . . . 5
III. DNA Glycosylases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
IV. Downstream BER Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
V. Mammalian BER . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
VI. Roles of BER Enzymes in Other Processes . . . . . . . . . . . . . . . . . 27
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28
Nucleotide Excision Repair in E. Coli and Man
Aziz Sancar and Joyce T. Reardon
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
II. Damage Recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
III. Mechanism of Excision Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
IV. Transcription-Coupled Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
V. Repair of Chromatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
VI. Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Photolyase and Cryptochrome Blue-Light Photoreceptors
Aziz Sancar
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
II. Phylogenetics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
III. Structure of Photolyase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
IV. Reaction Mechanism of Photolyase . . . . . . . . . . . . . . . . . . . . . . . 79
V. (6–4) Photolyase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
VI. Cryptochrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
v
vi CONTENTS
VII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
Coordination of Repair, Checkpoint, and Cell Death Responses

to DNA Damage
Jean Y. J. Wang and Sarah K. Cho
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
II. Overview of Biological Responses to DNA Damage. . . . . . . . . . 104
III. Molecular Components for the Initiation of DNA
Damage Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
IV. Apoptotic Effectors in DNA Damage Response . . . . . . . . . . . . . 115
V. DNA Repair Proteins in Damage Signaling . . . . . . . . . . . . . . . . 120
VI. Alternative Models for the Temporal Coordination of
DNA Damage Responses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
VII. Future Prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Functions of DNA Polymerases
Katarzyna Bebenek and Thomas A. Kunkel
I. DNA Polymerase Families . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

II. Structures and Compositions of DNA Polymerases . . . . . . . . . . 139
III. Functions of DNA Polymerases . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
IV. Polymerases for DNA Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
V. Polymerases for Replicating Undamaged DNA . . . . . . . . . . . . . 150
VI. Polymerases for Sister Chromatid Cohesion. . . . . . . . . . . . . . . . 151
VII. Mitochondrial DNA Replication . . . . . . . . . . . . . . . . . . . . . . . . . . 152
VIII. Polymerases for Replicating Damaged DNA . . . . . . . . . . . . . . . . 152
IX. Polymerases and Cell-Cycle Checkpoints. . . . . . . . . . . . . . . . . . . 155
X. Polymerases for Replication Restart and
Homologous Recombination . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
XI. Polymerases for DNA Mismatch Repair . . . . . . . . . . . . . . . . . . . . 156
XII. Polymerases in the Development of the Immune System . . . . 156
XIII. Biological Consequences of Polymerase Dysfunction . . . . . . . . 157
XIV. Closing Comments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
CONTENTS vii
Cellular Functions of DNA Polymerase and Rev1 Protein
Christopher W. Lawrence
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
II. Enzymological Studies With Pol and Rev1p . . . . . . . . . . . . . . . 172
III. Genetic Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
IV. Processes Other than General Translesion Replication
that Employ Pol and Rev1p. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
V. Regulation of Pol and Rev1p and Interactions with
other Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
VI. Conclusions and Speculations . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
DNA Polymerases and
Alexandra Vaisman, Alan R. Lehmann, and Roger Woodgate
I.
Historical Perspective. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
II.
Identification of RAD30 and its Orthologs . . . . . . . . . . . . . . . . 206
III.Biochemical Properties of Pol and Pol . . . . . . . . . . . . . . . . . . 207
IV.Translesion Synthesis by Pol and Pol. . . . . . . . . . . . . . . . . . . . 210
V.Structure of the Catalytic Core of S. cerevisiae Pol . . . . . . . . . . 212
VI.Regulation and Localization of Pol and Pol . . . . . . . . . . . . . . 215
VII.Mutations in Pol in XP Variants. . . . . . . . . . . . . . . . . . . . . . . . . 217
VIII.Pols and and the Polymerase Switch: Interactions
with PCNA and Rev1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219
IX. Protection from Cellular Effects of DNA Damage . . . . . . . . . . 220
X. Roles of Pol and Pol in Somatic Hypermutation . . . . . . . . . . 221
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
Properties and Functions of Escherichia

Coli : Pol IV and Pol V
Robert P. Fuchs, Shingo Fujii, and JØro^ me Wagner
I. DNA Pol IV, the dinB Gene Product . . . . . . . . . . . . . . . . . . . . . . 230

II. DNA Polymerase V, the umuDC Gene Product . . . . . . . . . . . . . 248
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
viii CONTENTS
Mammalian Pol : Regulation of Its Expression and Lesion Substrates
Haruo Ohmori, Eiji Ohashi, and Tomoo Ogi
I. Structures of the Genes and Proteins . . . . . . . . . . . . . . . . . . . . . 265

II. Enzymatic Properties of Pol . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
III. Possible Mechanisms of TLS by Pol In Vivo . . . . . . . . . . . . . . . 274
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
DNA Postreplication Repair Modulated by Ubiquitination and Sumoylation
Landon Pastushok and Wei Xiao
I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279
II. DNA Postreplication Repair Prokaryotes. . . . . . . . . . . . . . . . . . . 280
III. DNA Postreplication Repair in Eukaryotes . . . . . . . . . . . . . . . . . 281
IV. Ubiquitination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283
V. Protein Conjugation in PRR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 286
VI. Postreplication Repair via Covalent Modifications of PCNA . . 292
VII. Functional Conservation of Eukaryotic Postreplication Repair 295
VIII. Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
IX. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
Somatic Hypermutation: A Mutational Panacea
Brigette Tippin, Phuong Pham, Ronda Bransteitter, and Myron F. Goodman
I. Generation of Antibody Diversity . . . . . . . . . . . . . . . . . . . . . . . . . 307

II. Somatic Hypermutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
III. Apobec Protein Family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
IV. Biochemical Perspective. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
AUTHOR INDEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337

SUBJECT INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
PREFACE
The topic of DNA repair, now firmly embedded in the larger field of
biological responses to DNA damage, is in an extraordinarily dynamic state
at this time. In particular, productive inroads are being made into the
central issue of how cells sense the presence of DNA damage and initiate
specific signals in response to genomic insult of one type or another. The
term ‘‘genomic insult’’ is deliberately used in deference to the more familiar
term ‘‘DNA damage’’ because it is becoming increasingly apparent that the
primary event that initiates biological responses to genomic insult may be
arrested DNA replication rather than DNA damage itself. This volume,
entitled DNA Repair and Replication, is thus timely and appropriately focused.
The book comprises eleven chapters contributed by experts in the field.
It opens with discussions of the primary modes of the repair of DNA base
damage in the strict biochemical sense, namely, base excision repair
(BER), enzymatic photoreactivation (EPR) of both cyclobutane pyrimi-
dine dimers and [6–4] photoproducts, nucleotide excision repair (NER),
mismatch repair (MMR), and the repair of DNA double-strand breaks
(DSBR).
BER comprises a fundamental group of biochemical reactions that
notably processes many types of spontaneous base damage to DNA,
especially the damage generated by the pervasive reactive oxygen species
that continually swamp the intracellular milieu. Fromme and Verdine
provide a comprehensive discussion of these reactions, with an emphasis
on BER in mammalian cells. They also discuss the possible novel roles of
BER proteins in sensing damage and initiating checkpoint responses. The
chapter on NER contributed by Aziz Sancar and Joyce Reardon provide a
historical introduction to the biological response to DNA damage that is
evoked primarily by environmental reagents reactive with DNA and is now
known to comprise both transcription-coupled and transcription-inde-
pendent pathways. Its links to human hereditary diseases are also
discussed, as is the important and still murky area of chromatin
remodeling during NER. This phenomenon has long been intuitively
considered indispensable for the process, since the NER machinery is
physically massive once fully assembled, and it is not obvious how it could
access sites of base damage without some sort of chromatin modification.
Aziz Sancar has provided a comprehensive survey of EPR including
the phylogenetic relationship between DNA photolyases and other
light-absorbing proteins, notably those involved in circadian rhythm.
Unfortunately, this volume was not able to include chapters on other
ix
x PREFACE
aspects of DNA repair, such as mismatch repair and the repair of DNA
strand breaks.
Jean Wang and Sarah Cho have effectively tackled the formidable task of
attempting to integrate the coordination of DNA damage sensing and the
elaboration of checkpoint signals, a rapidly evolving and complex area of
contemporary research in biological responsiveness to genomic insult.
Sustained arrested DNA replication is lethal to cells. Both prokaryotic
and eukaryotic cells react to this threat with a series of diverse biological
responses that are designed to relieve the arrest while tolerating the
presence of the offending damage. Such damage can presumably be
repaired once the replicative crisis is resolved, although it remains unclear
why lesions at sites of replicative arrest are apparently refractory to repair.
Some of these responses promote the resumption of high-fidelity
semiconservative DNA synthesis without incurring mutations. Others,
presumably employed as a last-ditch response to arrested replication,
support DNA synthesis across sites of template DNA damage with a high
probability of mutation. Indeed, this phenomenon may constitute a
primary source of spontaneous mutagenesis in many organisms. Gratify-
ingly, half of this volume is dedicated to a discussion of these so-called
DNA damage–tolerance mechanisms, for much remains to be learned
about how cells determine which of the multiple responses to arrested
DNA replication to activate, and in what order.
Our understanding of error-prone (mutagenic) responses to arrested
DNA replication has experienced a flowering in the last 5 years because of
the discovery that prokaryotes, and especially eukaryotes, are endowed
with multiple specialized DNA polymerases that are able to effect
extension of DNA primer strands across sites of damage that cause the
arrest of high-fidelity DNA synthesis. It is now well appreciated that the
fundamental property of these specialized DNA polymerases that
promotes their ability to support so-called translesion DNA synthesis is a
dramatically reduced fidelity for nucleotide incorporation. This property
necessarily carries the risk of mutational catastrophe if such enzymes gain
access to stretches of undamaged DNA, and much remains to be learned
about how the regulation of such access is controlled in cells. A series of
chapters by Katarzyna Bebenek and Thomas Kunkel; Christopher
Lawrence; Alexandra Vaisman, Alan Lehmann, and Roger Woodgate;
Robert Fuchs, Shingo Fujii and Jérôme Wagner; Haruo Ohmori, Eiji
Ohashi, and Tomoo Ogi present current information on the plethora of
specialized polymerases in prokaryotes and eukaryotes. These chapters
include intriguing hints how critical switching events between high-fidelity
and low-fidelity polymerases may transpire during translesion DNA
synthesis.
PREFACE xi
Error-free mechanisms for DNA damage tolerance have been variously

dubbed postreplication repair, postreplicative recombinational repair,
repair by gap-filling, and replication fork regression, to name a few terms.
Landon Pastushok and Wei Xiao have provided a useful summary of the
important regulatory roles of specific types of posttranslational modifica-
tion of certain proteins, notably monoubiquitination and sumoylation, in
these biological responses, with an appropriate focus on the yeast
Saccharomyces cerevisiae. The enormous utility of the genetic versatility of
this lower eukaryotic is once again becoming a boon to the field.
In recent years, studies from several laboratories have generated
suggestive solutions to the long-standing mystery of how somatic
hypermutation transpires in immunoglobulin genes during antibody
maturation. Intriguingly, various familiar players on the DNA repair stage
appear to be critical, including the specialized DNA polymerases just
mentioned. In this volume, Myron Goodman and his colleagues have
provided a cogent summary of class switch recombination and somatic
hypermutation that incorporates the known role of activation-induced
cytosine deaminase (AID), as well as the possible roles of error-prone DNA
polymerases, base excision repair, and mismatch repair.
The field of biological responses to DNA damage is grossly underserved
with contemporary texts that address current problems and perspectives.
Wei Yang and her publisher, Elsevier, are to be congratulated on their
timely elaboration of this comprehensive volume. This collection of
reviews will serve well not only the DNA repair community, but also
students of DNA metabolism and cell regulation in general.
Errol C. Friedberg
Laboratory of Molecular Pathology
Department of Pathology
University of Texas Southwestern Medical Center
Dallas, TX 75390–9072
BASE EXCISION REPAIR
By J. CHRISTOPHER FROMME* AND GREGORY L. VERDINE*,À
*Department of Molecular and Cellular Biology, Harvard University,

Cambridge, Massachusetts, 02138
ÀDepartment of Chemistry and Chemical Biology, Harvard University,
Cambridge, Massachusetts, 02138
I. Introduction . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 1
II. Types of Damage Repaired by BER . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 5
III. DNA Glycosylases. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 7
A. Mechanistic Classes . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 7
B. Distinct Structural Classes. .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 9
C. Damage Recognition and Searching . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 10
D. Catalysis . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 16
IV. Downstream BER Enzymes . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 18
A. AP Endonucleases. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 18
B. Short-Patch versus Long-Patch Repair . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 19
V. Mammalian BER . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 23
A. Additional Components . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 23
B. Knockout Mice and the Role of BER Proteins in Human Disease . . . .. . . . . . 24
VI. Roles of BER Enzymes in Other Processes . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 27
A. BER-Like Enzymes in Plant Development . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 27
B. Thymine DNA Glycosylase .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 28
References. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 28
I. Introduction
When Marshall McLuhan coined the phrase ‘‘the medium is the mes-
sage,’’ he could not have imagined just how perfectly his shrewd commen-
tary on pop culture would summarize the entire molecular underpinnings
of genetics. The medium of heredity—the covalent structure of the nu-
cleobases in DNA and RNA—directly provides the information content for
all genetic transactions: Change the covalent structure, change the biolog-
ic meaning. It thus comes as no surprise that living systems expend
considerable energy in an ongoing struggle against spontaneous genetic
change. Uncovering the origins of such change and the nature of the
defense against it is of broad significance for understanding issues ranging
from evolution to carcinogenesis.
The most wide-ranging threat to genetic integrity is posed by the attack
of environmental agents on DNA nucleobases, resulting in their sponta-
neous covalent modification (Lindahl, 1993). Nucleobases are subject to
the attack of oxidants, alkylating agents, ultraviolet light, and other forms
of electromagnetic radiation; even the solvent water that endows DNA with
1 Copyright 2004, Elsevier Inc.

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2 FROMME AND VERDINE
its miraculous biologic properties can cause hydrolytic damage to DNA

bases. Nearly all of the damaged bases resulting from chemical modifica-
tion, often referred to as ‘‘lesions,’’ interfere with the template function of
DNA, thereby giving rise to point mutations (Lindahl, 1993) or aberrant
transcripts (Bregeon et al., 2003). In certain cases, the lesions possess
unstable glycosidic bonds and therefore undergo further spontaneous
conversion to abasic sites (Stivers and Jiang, 2003), which are themselves
highly genotoxic (Loeb and Preston, 1986).
The negative selective pressure imposed by lesion nucleobases has driven
the evolution of repair pathways dedicated to recognition and removal
of base lesions, followed by restoration of the original DNA sequence-
(Lindahl, 1993). The major pathway for this type of repair (Fig. 1) is
initiated by the excision of a damaged base and is therefore known as base
excision DNA repair (BER). Another repair pathway, nucleotide excision
repair (NER), can also repair some lesion nucleobases that severely distort
the DNA helix. The NER pathway is discussed in Chapter 2.
The enzymes responsible for catalyzing the initiation of BER are desig-
nated as DNA glycosylases, because they catalyze cleavage of the glycosidic
bond linking a lesion nucleobase to the DNA backbone. DNA glycosylases
are specialists, each recognizing one lesion, or at most a small subset of
lesions; most organisms express multiple enzymes to gain broad protec-
tion from various kinds of routinely encountered genetic insults. Locating
these damaged bases embedded in a more than million-fold excess of
normal DNA represents a formidable biologic version of the ‘‘needle in
the haystack’’ problem. DNA glycosylases channel the chemically diverse
universe of lesion bases into a small number of products suitable for
processing by downstream enzymes in the BER pathway.
DNA glycosylases fall into two mechanistic classes: monofunctional DNA
glycosylases displace the lesion base using a molecule of solvent water,
whereas bifunctional DNA glycosylases (glycosylase/lyases) displace the
lesion base by using a nucleophilic active site residue on the protein, which
is always an amine (Fig. 2). The covalently linked enzyme/DNA intermedi-
ate thus formed undergoes a cascade of further reactions, ultimately
resulting in cleavage of the DNA backbone on the 30 -side of the lesion base.
After a DNA glycosylase excises a lesion base, the resulting product
is further processed by an AP (apurinic/apyrimidinic) endonuclease,
which nicks the DNA backbone on the 50 side of the lesion base. When
an AP endonuclease acts on an abasic site generated by a monofunctional
DNA glycosylase, the product is a 50 -deoxyribosephosphate (50 -dRP) group
(Fig. 3). This species is usually removed by DNA polymerase , the repair
polymerase. However, an alternative pathway is sometimes used in which
DNA polymerase /", together with PCNA and FEN-1, displaces the strand
BASE EXCISION REPAIR 3
Fig. 1. Base excision repair pathway for repair of DNA base damage. Base damage is
indicated by the color red and an asterisk. The key enzymatic activities are indicated at
each step, but some steps require proteins not indicated. ‘‘Pol’’ is an abbreviation for
‘‘polymerase.’’ (See Color Insert.)
on the 30 side of the nick. This ‘‘long-patch’’ repair process is thought to

be important for replication-associated repair of base damage. In contrast,
when AP endonuclease acts on the product generated by a bifunctional
glycosylase, the entire lesion nucleoside is removed, and repair polymeri-
zation can proceed directly. Finally, DNA ligase covalently reseals the DNA
backbone.
4
FROMME AND VERDINE
Fig. 2. Mechanistic scheme of the reactions catalyzed by DNA glycosylases. DNA glycosylases must first recognize and bind to
nucleobase damage (1). The monofunctional DNA glycosylases, such as uracil DNA glycolase and MutY, then catalyze one-step removal
of the lesion base to generate an abasic site (4). Bifunctional DNA glycosylases, such as hOGG1, MutM, and EndoIII, catalyze additional
reactions via attachment of an active-site nucleophilic moiety (Nu) (2), resulting in 30 - (3, in the case of glycosylases with a primary
amine nucleophile) or 30 - and 50 - (6, in the case of glycosylases with a secondary amine nucleophile) nicking of the DNA strand. A
characteristic of the Schiff base intermediate (2) is that it can be intercepted by the reducing agent sodium borohydride (NaBH4) to
generate a stable covalent enzyme-DNA complex (5). This figure is reprinted with permission (Fromme et al., 2004b).
Fig. 3. Reactions catalyzed by apurinic/apyrimidinic (AP) endonucleases. An AP

endonuclease will generate a 50 -dRP group and a nicked strand when acting on an
abasic site. Alternatively, a single-nucleotide gap will be created if the substrate is the
-elimination product of a bifunctional DNA glycosylase.
II. Types of Damage Repaired by BER

As mentioned above, a wide variety of damaged DNA bases result from the
attack of various environmental agents (Table I). The enormous molar
excess of water (55 M) over the genome provides driving force for water
to cause hydrolytic deamination of amine-containing DNA bases. The
Table I
Substrates of Base Excision Repair
Source of damage Example lesion Responsible DNA glycosylases
Water/alkaline Uracil UDG, MUG, SMUG

Oxidation 8-oxoguanine OGG1, MutM
Alkylation 3-methyladenine MagI, AlkA, MagIII, AAG
Ultraviolet light Thymine dimer T4 endonuclease V
predominant target of this reaction is cytosine, which undergoes conversion

to uracil (Lindahl, 1993). Uracil residues in DNA are an efficient substrate
for BER catalyzed by uracil DNA glycosylases, of which several distinct forms
are known. Interestingly, hydrolytic deamination of 5-methylcytosine gen-
erates the normal base thymine, but this is recognized as a BER substrate by
its being paired opposite G instead of A. Hydrolytic deamination of adenine
generates hypoxanthine, another BER substrate (Stivers and Jiang, 2003).
The by-products of cellular respiration include several ferociously
reactive oxygen species (ROS) such as hydroxyl radical, superoxide radical,
and hydrogen peroxide. ROS are also found at high levels in cigarette
smoke. ROS can react with many components of the cell, including
DNA bases. Of the four canonical DNA bases, guanine is the most sensitive to
oxidation (Burrows and Muller, 1998). The oxidation product,
8-oxoguanine (oxoG), is quite stable in DNA (Cullis et al., 1996) and is a
potent mutagen owing to its overwhelming preference for mispairing with
adenine during DNA replication (Grollman and Moriya, 1993; Shibutani
et al., 1991). BER is the primary pathway for repair of oxoG lesions. Other
known oxidative lesions include thymine glycol, 5-hydroxycytosine, and
formamidopyrimidine (fapy) (Lu et al., 2001).
Many exogenous mutagens are alkylation agents that express their
toxicity through covalent attachment to DNA bases. Alkylation of a DNA
base can lead to mispairing or result in a lesion base with a particularly
weak glycosidic bond, increasing the likelihood of depurination. There are
several known alkylation-damage products, including 7-methylguanine and
3-methyladenine, which are repaired by BER (Wyatt et al., 1999). Some
alkylated bases are repaired by systems other than BER; for instance, the
alkylation product O6-methylguanine is repaired by direct reversion (Foote
et al., 1980; Myrnes et al., 1982; Olsson and Lindahl, 1980). Recently, a novel
dioxygenase mechanism for direct reversion of alkylated bases has been
observed in catalysis by AlkB (Sedgwick, 2004).
The most well-known form of ultraviolet light–induced base damage is
the pyrimidine dimer. Although the hallmark repair system for this type of
DNA lesion is NER, the BER response is also known to process cyclobutane
thymine dimers (Seawell et al., 1980).
III. DNA Glycosylases
A. Mechanistic Classes
There are two distinct mechanistic classes of DNA glycosylases. The
monofunctional glycosylases remove lesion bases from the DNA backbone
by cleavage of the glycosidic bond, displacing the lesion base using hy-
droxide derived from water. The product of this reaction is an abasic site
(Dodson et al., 1994; Sun et al., 1995) (Fig. 2, structure 4), which is the
primary substrate for AP endonuclease (Mol et al., 2000a; Wilson and
Barsky, 2001). Bifunctional glycosylases remove lesion bases via displace-
ment with a nucleophilic active-site residue (Dodson et al., 1994; Sun et al.,
1995; Weiss and Grossman, 1987). Dependent on the structural family of
bifunctional glycosylase, the nucleophile is the primary amine of an
internal lysine residue (Dodson et al., 1994; Kuo et al., 1992; Nash et al.,
1997; Sun et al., 1995; Thayer et al., 1995), the secondary amine of an N-
terminal proline residue (Tchou and Grollman, 1995; Zharkov et al.,
1997), or the primary amine of a N-terminal threonine residue (Dodson
et al., 1993). Bifunctional glycosylases, by virtue of the covalent attachment
they form with the lesion nucleoside (Fig. 2, structure 2), catalyze addi-
tional transformation of the substrate DNA, resulting in a nicked phos-
phate backbone (Dodson et al., 1994). They also perform this strand–
nicking reaction (sometimes referred to as ‘‘AP lyase’’ activity) on abasic
site substrates (McCullough et al., 2001). Depending on the nature of the
active-site amine, the product of strand nicking may be singly (glycosylases
with a primary amine nucleophile) or doubly (glycosylases with a second-
ary amine nucleophile) nicked (Fig. 2, structures 3 and 6). The strand
nicking occurs via a -elimination (Bailly and Verly, 1987; Kow and
Wallace, 1987; Mazumder et al., 1991) (singly nicked) or , -elimination
(doubly nicked) mechanism (Bhagwat and Gerlt, 1996).
An experimentally useful feature of the bifunctional mechanism is
the capability of reducing agent (i.e., NaBH4) to intercept the covalent
enzyme-DNA intermediate (Dodson et al., 1994; Nash et al., 1996; Sun et al.,
1995). The result of reduction is a covalent single bond between the
glycosylase and substrate DNA that is quite stable (Fig. 2, structure 5).
This ‘‘borohydride trapping’’ procedure has been useful in the isolation
(Bruner et al., 1998; Nash et al., 1996; Piersen et al., 1995) and characteri-
zation (Girard et al., 1997; Ikeda et al., 1998; Nash et al., 1997; Sidorkina
Fig. 4. Structures of DNA glycosylase-DNA complexes, representing the five known

structural classes of DNA glycosylase. In each panel DNA is shown as gold sticks with the
and Laval, 2000; Sun et al., 1995; Zharkov et al., 1997) of numerous
bifunctional DNA glycosylases. DNA glycosylase catalytic mechanisms are
discussed in further detail below.
B. Distinct Structural Classes

There are several different structural classes of DNA glycosylases (Fig. 4),
but the boundaries of these classes do not strictly coincide with those of the
mechanistic classes. The UDG structural family includes the DNA glyco-
sylases (UDG or UNG), MUG, TDG, and SMUG, all monofunctional
glycosylases. These proteins contain a single domain and recognize uracil
or thymine mismatches in DNA. The MutM structural family includes the
two-domain bifunctional glycosylases MutM (also known as 8-oxoguanine
glycosylase or Fpg) and endonuclease VIII (also known as Nei), which
repair various types of oxidative damage. Until recently it was believed that
MutM family members were found only in bacteria. This view changed
with the relatively recent discovery of several mammalian homologs, re-
ferred to as the NEIL (Nei-like) proteins (Bandaru et al., 2002; Hazra et al.,
2003; Morland et al., 2002; Takao et al., 2002a).
AAG (also known as ANPG), the only known member of its own
structural class, is a single-domain eukaryotic monofunctional glycosylase
that repairs alkylation damage (Engelward et al., 1993). Endonuclease V
from the T4 virus, which also has no known structural relatives, is a small
(16-kD) bifunctional glycosylase that repairs ultraviolet-induced thymine
dimers (Dodson et al., 1993).
The largest and most functionally diverse structural family of DNA
glycosylases is the HhH-GPD superfamily (Nash et al., 1996; Thayer et al.,
1995). The family includes both monofunctional and bifunctional DNA
glycosylases, and members recognize a variety of lesions arising from
oxidative, alkylation, and hydrolytic damage. Some members of both the
MutM and HhH-GPD families contain structural metal ions that play no
direct role in catalysis.
damage base(s) in red, and glycosylases are shown in ribbon format. (A) T4
endonuclease V bound to DNA containing a thymine dimer (Vassylyev et al., 1995).
(B) Uracil DNA glycosylase bound to pseudouridine-containing DNA (Parikh et al.,
2000). (C) Human 8-oxoguanine glycosylase 1 bound to DNA containing 8-oxoguanine
(Bruner et al., 2000). (D) Human alkyladenine glycosylase bound to DNA containing
ethenoadenine (Lau et al., 2000). (E) MutM (bacterial 8-oxoguanine glycosylase) bound
to DNA containing 8-oxoguanine (Fromme and Verdine, 2003a). (See Color Insert.)
C. Damage Recognition and Searching

One of the major challenges facing DNA glycosylases is locating
damaged bases embedded amid an overwhelmingly larger number of
normal bases. It is undesirable for DNA glycosylases to act on undamaged
DNA, if only because of the potential toxicity of their abasic-site products
(discussed later). To understand how glycosylases search for damage, we
must first understand the basis for recognition of damage. A particularly
informative avenue for revealing the atomic basis of damage selection is
structural biology. There has been a recent windfall of structural data
addressing the issue of damage recognition by DNA glycosylases (Fromme
et al., 2004b). The structure of a recognition complex, in which a DNA
glycosylase is bound to duplex DNA containing a lesion base, can be
especially informative. The caveat associated with any recognition complex
is that some perturbation is necessary to obtain the structure, either a
mutation of the enzyme or the use of a noncleavable substrate analog, so
that one is never viewing a true precatalytic state. However, if the pertur-
bations are subtle enough, it can be assumed that the structure obtained is
a reasonable facsimile of the true natural state.
1. Damage Recognition
Lesion base-containing recognition complexes for many DNA glycosy-
lases have now been structurally characterized (Table II). These include
uracil-DNA glycosylase (UDG), representing the first structure of a eukary-
otic DNA glycosylase-DNA complex (Parikh et al., 1998; Slupphaug et al.,
1996) and MUG (Barrett et al., 1998, 1999), both members of the same
structural family. These enzymes remove uracil from DNA, and MUG will
also remove thymine when paired opposite guanine. MUG recognizes its
substrate primarily by interacting with the guanine paired opposite the
substrate base, and it exhibits little selectivity within the active site. UDG
does not make any specific contacts with the guanine base and only makes
direct contact with uracil in the product complex. The authors suggest
that UDG substrate discrimination is mainly a result of the instability of
U:G pairs. Extensive work has been performed on the structural biology of
UDG, often with the goal of elucidating the catalytic mechanism (see
below). Structures of the uracil glycosylase SMUG1 in complex with
DNA have recently been obtained (Wibley et al., 2003). Unfortunately, it
was not possible to obtain a true recognition complex with SMUG1, so the
details of uracil recognition by that enzyme remain to be determined.
Recognition complexes are also available for T4 endonuclease V (Vassylyev
et al., 1995), hAAG (Lau et al., 2000), MutM (Fromme and Verdine,
2003a), and the HhH-GPD glycosylases endonuclease III ( J. C. Fromme
Table II
Structurally Characterized DNA Glycosylase-DNA Complexes
Mechanistic Structural
Glycosylase class class DNA-bound structures available
AAG Monofunctional AAG Recognition, abasic analog, and

mutant complexes
AlkA Monofunctional HhH-GPD Abasic analog complex
EndoIII Bifunctional HhH-GPD Recognition and trapped
intermediate complexes
EndoVIII Bifunctional MutM Trapped intermediate complex
OGG1 Bifunctional HhH-GPD Recognition, trapped intermediate,
and mutant complexes
MUG Monofunctional UDG Product, substrate analog, and
nonspecific complexes
MutM Bifunctional MutM Recognition, trapped intermediate,
product, and abasic analog
complexes
MutY Monofunctional HhH-GPD Recognition and product-like
complexes
SMUG1 Monofunctional UDG Product and nonspecific complexes
T4 EndoV Bifunctional T4 EndoV Recognition complex
UDG Monofunctional UDG Product, substrate analog, and
mutant complexes
and G. L. Verdine, unpublished), hOGG1 (Bruner et al., 2000), and MutY

(Fromme et al., 2004a). T4 endonuclease V, the first DNA glycosylase for
which the structure of a lesion-containing DNA complex was determined,
is unique among DNA glycosylases by virtue of its intrahelical recognition
and removal mode. Whereas all other DNA glycosylases extrude their
substrate base into an extrahelical active site pocket, T4 endonuclease V
instead extrudes one of the two adenine bases paired opposite the thy-
mine-dimer substrate to gain access to the lesion.
On the basis of a structural study, AAG was thought to select for its
diverse array of alkylated substrates in DNA by sensing their electron
deficiency via stacking with aromatic residues (Lau et al., 2000). More
recently, it has been suggested that AAG recognizes its substrates, includ-
ing the deamination product hypoxanthine, principally by excluding
normal bases (O’Brien and Ellenberger, 2003). AAG has little discrimina-
tion for the identity of the base paired opposite a lesion, and consequently
makes sparse contact with this base.
The two distinct oxoG-glycosylases, MutM (found in prokaryotes) and
OGG1 (found in eukaryotes), recognize the oxoG:C pair in DNA some-
what differently (Fig. 5). OGG1 binds to oxoG in the anti glycosidic
Fig. 5. Comparison of 8-oxoguanine binding to the active sites of two different

8-oxoguanine glycosylases. The oxoG base is shown in red, DNA in gold, and enzyme
residues in cyan. (A) hOGG1 active site (Bruner et al., 2000). (B) MutM active site
(Fromme and Verdine, 2003a). Both panels used with permission. (See Color Insert.)
conformation, makes contact with the Watson–Crick face of oxoG using a

glutamine sidechain, and recognizes the oxidation product by hydrogen
bonding to the N7 proton of oxoG with the main-chain carbonyl oxygen of
a glycine residue (Bruner et al., 2000). MutM binds to oxoG in the syn
glycosidic conformation, makes contacts with the Watson–Crick face using
threonine and glutamate sidechains, and recognizes the oxidation status

of oxoG via hydrogen bonding to the N7 position with the main-chain
carbonyl oxygen of a serine residue (Fromme and Verdine, 2003a). The
strategy for cytosine recognition differs between the two enzymes, with
OGG1 employing two arginine residues and MutM using a single arginine
residue to contact the Watson–Crick face of cytosine.
Substrates of the structurally related enzymes endonuclease III and
MutY share one aspect in common: the base paired opposite the lesion
has a guanine-like Watson–Crick face. In the case of endonuclease III, the
preferred substrates are oxidized pyrimidines when paired opposite gua-
nine (though lesions paired opposite adenine are also processed). In the
case of MutY, the ‘‘lesion’’ substrate is adenine when paired opposite
oxoG. Both enzymes recognize the guanine-like Watson–Crick face of
the opposite base using two main-chain carbonyl oxygens (Fromme and
Verdine, 2003b; Fromme et al., 2004a). However, MutY possesses an addi-
tional C-terminal domain that has been demonstrated to be essential for
proper functioning of MutY (Chmiel et al., 2001; Li et al., 2000; Noll et al.,
1999). Interestingly, this domain shares low structural homology with the
MutT enzyme, an oxo-dGTP triphosphatase. Using data from a fluores-
cence-based study, it was initially proposed that MutY would extrude not
only the substrate adenine but also the partner oxoG from the DNA helix
(Bernards et al., 2002). This ‘‘double-flipping’’ mechanism was appealing
for several reasons, but the experimental evidence used to reach this
conclusion was unable to distinguish between the effects of DNA bending
and base extrusion. In fact, the crystal structure of MutY bound to duplex
DNA containing an oxoG:A pair reveals that the oxoG remains intrahelical
in the recognition complex (Fromme et al., 2004a) (Fig. 6). An interesting
aspect of the MutY oxoG recognition mode is that oxoG is bound in the
anti conformation, in contrast to the syn conformation oxoG normally
adopts when paired with adenine in DNA (Kouchakdjian et al., 1991;
McAuley-Hecht et al., 1994). The anti conformation forces the substrate
adenine into the active site, and the syn conformation may serve as a
homing mechanism for MutY while searching for damage (see following).
Recognition of adenine by MutY seems to occur mainly by exclusion of
cytosine, the other possible partner to oxoG. Endonuclease III can process
a variety of lesions, which is borne out in the structure of a dihydrouracil-
containing recognition complex, evidenced by a lack of direct interactions
between the enzyme and substrate base ( J. C. Fromme and G. L. Verdine,
unpublished results).
Structures that lack a bound DNA duplex, but contain a substrate lesion
in the free-base form, can also be informative. The nuclear magnetic
resonance structures of 3-methyladenine glycosylase I (known as MagI or
Fig. 6. Structure of MutY bound to DNA containing an A:oxoG pair. The DNA is
shown in gold, with oxoG in magenta and the substrate adenine in purple. MutY
extrudes the adenine base from the duplex, but the oxoG base remains intrahelical.
The [4Fe-4S] cluster is shown as yellow and orange spheres. Reprinted with permission
(Fromme et al., 2004a). (See Color Insert.)
Tag) with bound substrate analogs and accompanying biochemistry show

that MagI recognizes its substrate bases primarily through van der Waals
interactions and hydrogen bonds with the Watson–Crick and major-groove
face of the lesions (Cao et al., 2003). Crystal structures of 3-methyladenine
glycosylase III (MagIII) bound to substrate analogs, together with kinetic
analysis, demonstrate that MagIII recognizes its substrate via aromatic
stacking interactions and steric exclusion (Eichman et al., 2003).
Other crystal structures that hint at substrate recognition mechanism—
despite the absence of a lesion base—include the complex between the
bacterial 3-methyladenine glycosylase AlkA (MagII) and DNA containing
an abasic site analog (Hollis et al., 2000) and a trapped-intermediate
complex of endonuclease VIII (Zharkov et al., 2002). Both structures
establish the overall DNA binding mode but lack a concrete depiction
of substrate base recognition.
Several different DNA glycosylases are known to act on T:G mismatches
in DNA. The primary source of these mismatches is deamination of
5-methylcytosine (Duncan and Miller, 1980; Sved and Bird, 1990).
5-Methylcytosine is found in eukaryotes as an epigenetic modifier of
chromatin structure and transcription, and in prokaryotes as the basis
for self-protection from endogenous restriction enzymes. The human
MBD4 protein is of particular interest among these evolutionarily wide-

spread proteins. It possesses two domains, a methyl-CpG binding domain
and a T:G glycosylase domain. The thymine glycosylase activity of the
second domain is sequence specific, preferring substrates with a methy-
lated CpG site paired opposite the substrate thymine base (Hendrich et al.,
1999). The crystal structure of the glycosylase domain has been deter-
mined in the absence of substrate (Wu et al., 2003), confirming its
place within the HhH-GPD superfamily. The bacterial thymine DNA
glycosylase Mig is also a member of the HhH-GPD superfamily. The crystal
structure of Mig has been determined (Mol et al., 2002) but also lacks
substrate. Both of these enzymes have the potential to teach us new
substrate recognition mechanisms, but we are awaiting structures obtained
in the presence of lesion-containing DNA.
2. Searching for Damage

There are several competing theories concerning the nature of the
damage search process. The most basic mechanism involves a simple
three-dimensional diffusion search, wherein encounters between the
enzyme and DNA are random and transient, unless damage is located
during the encounter. Based on the steady-state frequency of oxoG-lesions
in the genome—about two oxoG residues per million guanines (ESCODD,
2002) or 1500 in a human cell—this method seems unlikely. A more
expedient mechanism invokes a one-dimensional search process, in which
the DNA glycosylase slides along the DNA duplex, thus greatly improving
the likelihood of finding damage. Within this latter mechanism, there is
room for variant methods of detecting DNA damage. It has been sug-
gested that DNA glycosylases that act on non-helix-destabilizing lesions
must extrude every base they encounter to recognize damaged bases
(Verdine and Bruner, 1997).
In contrast, DNA glycosylases that act on helix-destabilizing lesions
may home in on damaged bases indirectly, simply by binding preferen-
tially to deformable sites in DNA. The structure of a MutY-DNA com-
plex indicates a possible avenue for direct detection (Fromme et al.,
2004a). The syn configuration oxoG adopts when paired with adenine
projects the N1, N2, and O6 atoms of oxoG out of the major groove.
This protuberance interrupts an otherwise relatively smooth major
groove surface, and it is possible that the projection is directly detected
by MutY. This mechanism is satisfying in part because it removes the
possibility of MutY stalling at oxoG:C pairs, in which oxoG adopts the anti
conformation.
Using duplex DNA with multiple substrates separated by defined dis-
tances, ‘‘processivity’’ has been demonstrated for several DNA glycosylases
(Francis and David, 2003; Higley and Lloyd, 1993). However, it should be
noted that these studies do not use statistical arguments to distinguish
between true processivity and proximity bias. A recent study on MutY
demonstrated that its [4Fe-4S] cluster is redox-active when the enzyme is
DNA-bound (Boon et al., 2003), whereas it was previously believed to be
redox-inert. Furthermore, the redox state of the metal cluster modulated
the affinity of MutY for DNA. The authors of this study suggest the cluster
may play a role in damage searching, insofar as oxidative lesions prevent
transmission of electrons through DNA.
MYH is known to associate with PCNA, a protein complex essential for
DNA replication (Chang and Lu, 2002; Parker et al., 2001), and it has been
shown to associate with replication forks (Boldogh et al., 2001). This
colocalization may simplify MutY damage searching.
In eukaryotic organisms, DNA exists in a chromatin-bound state, with
accessibility related to transcriptional status. It seems likely that the
chromatin state will influence the ability of BER enzymes to search for
and repair damage. Indeed a recent study has shown that nucleosome-
bound DNA can be acted on by UDG and AP endonuclease, but not by
DNA polymerase (Beard et al., 2003). These results indicate that
chromatin remodeling is necessary for completion of the BER process,
but not for the recognition of damage. However, in another study,
polymerase was active upon nucleosome-bound substrates (Nilsen et al.,
2002), though it is possible that the nucleosomal structures were more
loosely packed in this study. Further efforts are necessary to establish the
unique requirements of BER on a chromatin substrate.
D. Catalysis
DNA glycosylases are interesting subjects for enzymological studies,
if only because their substrate is the genome. Bifunctional glycosylases
are especially interesting from a mechanistic standpoint because they
catalyze several sequential reactions within a single active site. The base
removal, or excision, step is shared by both mechanistic classes, and it is
likely catalyzed similarly by both. Catalytic studies of DNA glycosylases are
frequently complicated by several issues. One problem is that many glyco-
sylases are severely end-product inhibited, rendering multiple-turnover
kinetic studies uninformative. A second problem is that substrate binding
occurs in several time-consuming steps, as a result of the need for gross
structural rearrangement of the DNA duplex ( Jiang and Stivers, 2002).
Thus, the rates of Michaelis complex formation may be slower than those
of the chemical steps, making it difficult to ascribe rate constants to
specific events.
The mechanism of catalysis by UDG, including events from the initial

DNA binding process through nucleoside extrusion and uracil excision,
has been studied extensively by Tainer, Stivers, and their colleagues
(Parikh et al., 2000; Stivers and Drohat, 2001). Some of the highlights of
this research include the importance of a leucine residue for extrusion of
the uridine nucleoside from the helix, which can be mimicked by replace-
ment of the opposite guanine with a bulky pyrene residue ( Jiang et al.,
2002). Extrusion of uracil from the DNA duplex is the rate-limiting step in
catalysis by UDG ( Jiang and Stivers, 2002). Other studies have demon-
strated that uracil excision by UDG follows an SN1-type pathway (Dinner
et al., 2001; Werner and Stivers, 2000). The active site of UDG lowers the
pKa of the uracil leaving group by 3.4 units to facilitate catalysis (Drohat
and Stivers, 2000). Monofunctional and bifunctional glycosylases alike use
aspartate or glutamate sidechains to stabilize the partial positive charge
that is proposed to develop either on O40 or C10 in the transition state of
the excision reaction. An additional driving force for excision of uracil is
thought to derive from nearby phosphates in the DNA backbone (Dinner
et al., 2001; Jiang et al., 2003).
Alkylated lesion bases have especially labile glycosidic bonds, and there-
fore require less ‘‘powerful’’ catalysis by DNA glycosylases. Accordingly,
MagIII performs catalysis on alkylated substrates even when the conserved
catalytic aspartate is mutated (Eichman et al., 2003). However, the alkyl-
ation damage-specific glycosylase AlkA has been shown to be a sufficiently
powerful catalyst to remove normal bases from DNA (Berdal et al., 1998),
and AAG exhibits a 108-fold catalytic rate enhancement on the substrate
hypoxanthine (O’Brien and Ellenberger, 2003).
Monofunctional glycosylases replace the leaving group lesion base with
a water molecule. In contrast, bifunctional glycosylases substitute a nucle-
ophilic sidechain. The moieties used are an N-terminal proline in MutM
family members, an N-terminal threonine in T4 endonuclease V, and an
internal lysine in HhH-GPD glycosylases. The formation of iminium inter-
mediates, available only to bifunctional glycosylases, is what allows for the
subsequent steps of strand nicking through -elimination. As mentioned
above, the primary bifunctional intermediate can be ‘‘trapped’’ with a
reducing agent. There are now crystal structures of trapped intermediates
available from several glycosylases (Fromme et al., 2004b). One such
structure led to the discovery that hOGG1 uses the product of base
excision, the oxoG lesion base, as a cofactor to assist in catalysis of
-elimination (Fromme et al., 2003).
The catalytic mechanisms of DNA glycosylases, with special emphasis on
monofunctional glycosylases, is the subject of a recent review (Stivers and
Jiang, 2003).
IV. Downstream BER Enzymes
A. AP Endonucleases
The products of DNA glycosylase activity are quite similar despite the
varied nature of glycosylase substrates. All monofunctional DNA glycosy-
lases produce the same species: the abasic site. The abasic site produced
enzymatically is chemically identical to the abasic product of spontaneous
base loss from DNA. The abasic site exhibits considerable genotoxicity.
Not only do abasic sites lack the coding information necessary for tem-
plate-directed DNA synthesis but they can lead to stalled replication forks
(Higuchi et al., 2003; Loeb and Preston, 1986) and transcription bubbles
(Yu et al., 2003); events that are potentially mutagenic or even lethal. They
have also been shown to trap topoisomerase I through a covalent interac-
tion (Pourquier et al., 1997). Two studies by Guillet and Boiteux indicate
that, at least in yeast, the single major source of abasic sites in the genome
is from the action of UDG on uracil, and that these abasic sites are lethal in
the absence of AP endonuclease activity (Guillet and Boiteux, 2002, 2003).
Importantly, lethality was suppressed by deletion of UDG, but not by any
other DNA glycosylase examined.
AP endonucleases cleave the DNA strand on the 50 side of abasic sites,
resulting in a nick having 30 -OH and 50 -deoxyribosephosphate (dRP)
groups. In addition, AP endonucleases will process the -elimination
products of bifunctional DNA glycosylases by completely removing the
processed lesion nucleoside, leaving behind a free 30 -OH. AP endonu-
cleases also possess 30 -phosphoesterase activity, enabling them to remove
the 30 -phosphate group remaining after the ,-elimination activity of
some bifunctional glycosylases (Fig. 3).
The bacterium Escherichia coli possesses two different AP endonucleases,
exonuclease III and endonuclease IV (Ljungquist and Lindahl, 1977).
These enzymes define the two known structural classes of AP endonucle-
ase. The predominant enzyme in E. coli is exonuclease III, which has 30 !
50 exonuclease activity in addition to the characteristic AP endonuclease
activities detailed above (Rogers and Weiss, 1980; Weiss, 1976). Endonu-
clease IV, whose expression in E. coli is induced in response to oxidative
stress (Chan and Weiss, 1987), is homologous to the major AP endonucle-
ase found in baker’s yeast, APN1 (Popoff et al., 1990). The major human
AP endonuclease, APE1 (also known as Ref-1 or HAP1), is homologous to
exonuclease III (Demple et al., 1991). APE1 is multifunctional, possessing
redox-dependent transcriptional activation (Xanthoudakis et al., 1992)
and acetylation-dependent, redox-independent transcriptional repression
activities (Bhakat et al., 2003; Okazaki et al., 1994) in addition to AP
endonuclease activities (Fritz et al., 2003). It has also been suggested that
APE1 is the main proofreading enzyme for mistakes made by DNA
polymerase , based on its ability to remove mismatched bases at 30
termini (Chou and Cheng, 2002). The additional roles of APE1 will be
discussed in greater detail later.
Because AP endonuclease acts immediately downstream of the DNA
glycosylases, the DNA repair community has actively sought evidence of
interactions between the two classes of enzyme (Wilson and Kunkel, 2000).
Human APE1 has been shown to stimulate the activity of the human DNA
glycosylases UDG (Parikh et al., 1998), TDG (Waters et al., 1999), hOGG1
(Vidal et al., 2001b), and NTH1 (Marenstein et al., 2003), most likely
through competitive substrate binding, but perhaps through a more active
mechanism. The only demonstration to date of a direct physical interac-
tion between AP endonuclease and a DNA glycosylase is between the
human adenine glycosylase MYH and APE1 (Parker et al., 2001).
The structural biology of AP endonucleases has been well established
through the work of Tainer and colleagues (Mol et al., 2000a), with DNA-
bound structures available from structurally distinct human APE1 (Mol
et al., 2000b) and E. coli endonuclease IV (Hosfield et al., 1999). The DNA
cocrystal structures reveal how these enzymes use different folds to achieve
similar results (Fig. 7A and B). Both enzymes extrude the substrate abasic
site from the DNA helix in a similar manner to the DNA glycosylases,
bending the duplex in the process and inserting protein residues into the
minor groove. Both enzymes use bound metal ion(s) to catalyze strand-
nicking via hydrolysis, and each structure led to a plausible proposal for
the enzymatic mechanism. Notably, endonuclease IV appears to use three
zinc ions for catalysis.
A somewhat surprising aspect of the structure of the APE1-DNA com-
plex is the location of the two conserved cysteine residues responsible for
the redox activity of APE1. These residues—Cys65 and Cys93 (Walker et al.,
1993)—are not exposed to solvent but, instead, are buried within
the protein. This indicates that APE1 might undergo a significant
conformational change to carry out its redox activity (Fig. 7C).
B. Short-Patch versus Long-Patch Repair

After the action of AP endonuclease on an abasic site, DNA repair
polymerase (polymerase in eukaryotes or polymerase I in bacteria) can
remove the dRP group (so-called ‘‘dRPase activity’’) (Allinson et al., 2001).
This reaction is catalyzed by the N-terminal domain of polymerase via an
imine intermediate, analogous to the AP lyase activity of bifunctional
glycosylases (Piersen et al., 1996; Prasad et al., 1998). If this step
Fig. 7. The two structurally distinct AP endonucleases. (A) Human APE1, in blue,
bound to DNA containing a substrate abasic site, in red (Mol et al., 2000b).
(B) Endonuclease IV, in red, bound to its product DNA (Hosfield et al., 1999). The
abasic moiety of the dRP group is colored blue. (C) View of the APE1/DNA complex
highlighting the positions of the two cysteines involved in the redox activity of this
enzyme. The cysteine residues are yellow, seen buried within the interior of the protein.
(See Color Insert.)
transpires, the product is chemically identical to the gap resulting from AP

endonuclease activity on a bifunctional elimination product. The one
nucleotide gap is filled in by the polymerase domain of DNA polymerase
, and the strand is resealed by a DNA ligase III/XRCC1 complex (Cappelli
et al., 1997). DNA ligase I has also been implicated as the functional ligase
acting at this stage, based on its association with polymerase (Prasad et al.,
1996) and competence within an in vitro BER assay (Nicholl et al., 1997).
The final steps described above are referred to as ‘‘short-patch’’ base
excision repair, because of the single nucleotide polymerization needed
to complete the repair process (Fig. 1, bottom right).
An alternative repair process (Frosina et al., 1996; Matsumoto et al.,
1994), termed ‘‘long-patch’’ base excision repair (Fig. 1, bottom left),
occurs when DNA polymerase /" initiates polymerization from the free
30 -OH adjacent to the dRP group resulting from AP endonuclease activity.
The polymerization incorporates between 2 and 15 nucleotides, displacing
the strand containing the 50 -dRP (called the ‘‘flap’’) and requiring FEN-1,
a nuclease that removes this flap (Klungland and Lindahl, 1997). Long-
patch repair has been reconstituted in vitro (Matsumoto et al., 1999;
Pascucci et al., 1999) and was shown to require both PCNA and the
nuclease FEN-1.
If flap removal produces a gap of fewer than 6 nucleotides, polymerase
can be a key player in long-patch repair (Fortini et al., 1998; Wilson, 1998).
An in vitro reconstitution of long-patch repair was achieved using polymer-
ase and demonstrated that PARP-1 (poly ADP-ribose polymerase-1)
stimulated the long-patch activity of polymerase (Prasad et al., 2001).
However, another report indicated that PARP-1 actually slows down the
repair reaction (Allinson et al., 2003). PARP-1 binds to single-strand nicks
in DNA (Benjamin and Gill, 1980) and is believed to play a role in damage
repair by scanning the genome for these nicks. PARP-1 was shown to interact
with the product of AP endonuclease activity in an ultraviolet cross-linking
study (Lavrik et al., 2001). The role of PARP in BER is discussed further later.
Recent structural studies of complexes between FEN-1, PCNA, and DNA
reveal the basis for the flap specificity of FEN-1 (Chapados et al., 2004).
FEN-1 binds to a kinked structure of the DNA duplex made possible by the
discontinuity of the nicked strand. The authors use the structure of a FEN-
1 peptide bound to PCNA to propose a model of how PCNA orients FEN-1
for interaction with DNA at a replication site (Fig. 8). Furthermore, it is
proposed that FEN-1 and the next downstream enzyme, DNA ligase, both
use similar features of nicked-DNA for substrate recognition while bound
to PCNA.
Physical interactions between APE1 and FEN-1 and APE1 and PCNA
have been demonstrated in copurification experiments (Dianova et al.,
Fig. 8. Modeled structure of a FEN-1/PCNA/DNA complex. This model was

generated using coordinates from structures of a FEN-1/DNA complex and a complex
between PCNA and a peptide fragment of FEN-1 (Chapados et al., 2004). PCNA is
shown in green, FEN-1 in blue, and DNA in gold. The DNA is presumed to extend
downward from its point of contact with FEN-1. (See Color Insert.)
2001). Together with the known interactions between FEN-1 and PCNA
(Wu et al., 1996), these interactions indicate that long-patch repair is a
tightly coordinated event, using interactions between enzymes and with
PCNA as a scaffold.
It has been noted that 50 -dRPs are more likely to provoke long-patch
repair than are single-nucleotide gaps (Klungland et al., 1999a). 50 -dRPs
arise after the sequential actions of a monofunctional glycosylase and AP
endonuclease, whereas single-nucleotide gaps arise from the actions of a
bifunctional glycosylase and AP endonuclease. Furthermore, bifunctional
glycosylases (OGG1, MutM, EndoIII, and EndoVIII) seem to be restricted
to oxidatively damaged substrates. What is unique about these substrates
that requires short-patch repair instead of long-patch repair? Lindahl
and colleagues have proposed that ionizing radiation, a major source of
oxidative lesions via generation of free radicals, tends to generate multiple

lesions in a concentrated area; long-patch repair might lead to a disastrous
double-strand break if two lesions lay on opposite strands within a few
basepairs of each other (Klungland et al., 1999a). Therefore, by using
bifunctional glycosylases to initiate repair at oxidative lesions, cells can
avoid the potentially harmful long-patch repair process.
V. Mammalian BER
A. Additional Components
BER in mammalian cells is more complex than in simpler eukaryotes
(Izumi et al., 2003; Memisoglu and Samson, 2000), involving interactions
with additional proteins like XRCC1 (Thompson and West, 2000) and p53
(Offer et al., 2001b) that coordinate repair and alert the cell to the
presence of damage. APE1 is suspected to be the key BER protein involved
in these extra layers of complexity (Fritz et al., 2003). The redox activity
of APE1 is known to regulate the DNA-binding affinity of several tran-
scription factors. The first example of such an interaction was with the
transcription factors Fos and Jun (Xanthoudakis et al., 1992), which
heterodimerize to form AP-1, an oxidation-sensitive transcription factor.
APE1, using two specific cysteine residues, reduces AP-1 in a thioredoxin-
dependent manner (Wei et al., 2000). The yeast version of APE1 does not
contain the conserved cysteine residues critical for redox function, lend-
ing credence to the view that this activity occurs only in higher eukaryotes
(Fritz et al., 2003).
APE1 also serves as a redox factor for p53 (Gaiddon et al., 1999;
Jayaraman et al., 1997), NF-B (Mitomo et al., 1994), HIF-1 (Huang et al.,
1996), and other transcription factors (Flaherty et al., 2001). The redox
activity of APE1 has been shown to be stimulated by phosphorylation by
casein kinase II and protein kinase C both in vitro and in vivo (Fritz and
Kaina, 1999; Hsieh et al., 2001), indicating that there may be a mechanism
whereby damage sensed by APE1 leads to its phosphorylation and
subsequent activation. This hypothesis is especially appealing considering
the roles of casein kinase II and protein kinase C in the DNA damage
response (Ghavidel and Schultz, 2001; Yoshida et al., 2003).
PARP-1 is a signaling enzyme that transfers ADP-ribose groups from
NADþ to itself and other nuclear proteins in response to detecting DNA
damage. Poly(ADP)ribosylation of histones has been shown to loosen
chromatin (de Murcia et al., 1986), indicating that damage detection by
PARP-1 leads to increased access to the site of damage for other repair
proteins. PARP-1 interacts with DNA polymerase in GST-pulldown assays

(Dantzer et al., 2000), and a knockout mouse model demonstrated the key
role this enzyme plays in BER (Dantzer et al., 1999), as demonstrated by
the inadequate repair of abasic sites by PARP-1-deficient cell extracts.
Interestingly, the interaction between PARP-1 and polymerase does
not depend on poly(ADP)ribosylation but does depend on the presence
of damaged DNA, whereas the interaction between PARP-1 and XRCC1 is
dependent on poly(ADP)ribosylation but independent of DNA (Dantzer
et al., 2000; Masson et al., 1998).
XRCC1 (x-ray repair cross-complementing protein 1) is a nonenzymatic
scaffolding protein that binds to other key BER enzymes, APE1 (Vidal et al.,
2001a), DNA polymerase (Caldecott et al., 1996; Kubota et al., 1996), and
DNA ligase III (Caldecott et al., 1994). More recently, XRCC1 has been
shown to interact with hOGG1 in both GST-pulldown and yeast two-hybrid
assays (Marsin et al., 2003). In addition, XRCC1 stimulates the repair
activity of hOGG1 on DNA containing oxoG, similar to stimulation by
APE1.
The tumor suppressor p53 plays a more direct role in BER in addition to
transcriptional regulation. p53 stimulates the rate of an in vitro BER system
by directly interacting with DNA polymerase and stabilizing the interac-
tion between the polymerase and abasic DNA (Zhou et al., 2001). The
in vivo relevance of this finding was validated when mutations in the trans-
activating domain of p53 failed to inactivate BER in mammalian cells,
whereas mutations elsewhere in p53 diminished BER (Offer et al., 2001a).
Another more recent study indicates that p53 can potentiate the forma-
tion of abasic sites by repressing transcription of 3-methyladenine DNA
glycosylase in response to genotoxic stress (Zurer et al., 2004). This mech-
anism might alleviate the mutational burden by ensuring that the level of
abasic site formation does not outpace the level of APE1.
XPG is an endonuclease associated with the NER pathway in eukaryotes.
The repair of oxidized lesions in vitro has been shown to be accelerated by
XPG, through interactions with the DNA glycosylase hNTH1 that increase
the affinity of hNTH1 for DNA (Klungland et al., 1999a).
B. Knockout Mice and the Role of BER Proteins in Human Disease

Given the importance of DNA glycosylases for BER, the DNA repair
community anxiously awaited the results of mouse knockout studies. The
initial results were generally greeted with disappointment, as animals
lacking particular DNA glycosylases seemed phenotypically normal. Early
studies of AAG (also known as ANPG)-knockout mice demonstrated an
increased sensitivity to methylating agents in cells derived from knockout
embryos, but the mice themselves exhibited no apparent phenotype

(Engelward et al., 1997). Parenthetically, the knockout studies were useful
in defining the in vivo substrate range for this glycosylase (Engelward et al.,
1997; Hang et al., 1997).
The generation of UDG (also known as UNG) knockout mice (Nilsen
et al., 2000) also revealed these animals to be outwardly normal despite an
increase in the steady-state genomic level of uracil. Cell extracts from
knockout animal tissue demonstrated a persistent uracil glycosylase activi-
ty, later definitively attributed to SMUG1 (Nilsen et al., 2001). These two
studies established the role of UDG during DNA replication and that of
SMUG1 as a constitutively expressed enzyme found only in higher eukary-
otes. A more recent study revealed that UDG knockout mice eventually
develop B cell lymphomas, highlighting the increasingly recognized im-
portance of UDG in the immune system (Nilsen et al., 2003). The role of
UDG in somatic hypermutation is discussed in Chapter 11.
OGG1 knockout mice appeared phenotypically normal but displayed
increased levels of oxoG in their genomes (Klungland et al., 1999b;
Minowa et al., 2000). Further studies showed that cells from knockout
mice were more susceptible to exogenous mutagens both in vitro and
in vivo (Arai et al., 2002, 2003). Knockout mouse embryo fibroblasts were
used to show that oxoG can be repaired independent of OGG1 in a
transcription-coupled repair process (Le Page et al., 2000). A recent study
has shown that OGG1 knockout mice exhibit a fivefold increase in lung
tumors at age 18 months (Sakumi et al., 2003), indicating that the resulting
mutational burden eventually manifests itself via carcinogenesis. Curious-
ly, in this same study it was shown that disruption of MTH1, which is
responsible for sanitizing the nucleotide precursor pool of oxo-dGTP by
catalyzing its hydrolysis to oxo-dGMP and pyrophosphate, suppressed the
tumorigenic phenotype of OGG1 deletion.
MBD4 knockout mice are viable but display increased mutation rates at
CpG sites (Millar et al., 2002). When these knockout mice where crossed
with mice predisposed to colon cancer, the tumorigenic potential of the
MBD4 deletion was revealed. A further study highlighted the important
role of MBD4 in triggering apoptosis, as a reduced apoptotic response was
observed in cells from knockout mice (Sansom et al., 2003).
NTH1 knockout mice, though otherwise viable, were crucial to the
discovery of novel mammalian homologs of Nei, the NEIL proteins (Elder
and Dianov, 2002; Ocampo et al., 2002; Takao et al., 2002b). The NEIL
family currently consists of three family members. Surprisingly, NEIL3
contains a C-terminal domain that is homologous to both topoisomerase
III and APE2, an AP endonuclease-like protein with unknown function
(Takao et al., 2002a).
It was initially assumed that functional redundancy obscured any obvi-

ous phenotypic effects in animals genetically engineered to lack certain
DNA glycosylases (Parsons and Elder, 2003). However, the recent results
with OGG1 and UDG knockout animals indicate that deficiency of a single
DNA glycosylase in some cases is sufficient to evoke carcinogenesis, but
this phenotype takes time to develop.
Although no MutY knockout animal is available yet, there is now com-
pelling genetic evidence that this DNA glycosylase acts as a tumor suppres-
sor in humans. The human homolog of MutY (adenine DNA glycosylase),
hMYH, has been persuasively linked to colon cancer. The first observation
of this fact came from a study of familial colon cancers not associated with
inherited deficiencies in the usual suspect, APC (Al-Tassan et al., 2002).
This and subsequent studies confirmed the prevalence of two specific
MYH polymorphisms in such cancers and showed that these polymorph-
isms are associated with tumorigenesis in a number of unrelated patients
(Cheadle and Sampson, 2003; Jones et al., 2002; Sampson et al., 2003;
Sieber et al., 2003). The effects of the polymorphisms on enzyme function
were shown using the equivalent mutations in the E. coli homolog (Chmiel
et al., 2003) and seem to stem mainly from reduced activity of the enzyme.
Furthermore, the reduction in activity was exacerbated at known ‘‘hot-
spot’’ sequences (Al-Tassan et al., 2002) mutated in the somatic APC genes
of patients inheriting defective MYH.
APE1 is overexpressed in many cancers (Holmes et al., 2002; Kakolyris
et al., 1997; Moore et al., 2000; Robertson et al., 2001) and is essential for
development (Xanthoudakis et al., 1996). A recent study found that
granzyme A cleaves APE1 as part of the cytotoxic T cell–initiated cascade
leading to cell death; overexpression of a noncleavable version of APE1
resulted in cells that were less sensitive to granzyme A-mediated cell death
(Fan et al., 2003). Granzyme A activity was seen to affect both the AP
endonuclease and redox functions of APE1, though redox activation of
transcription factors was suggested to be more critical for death avoidance.
The authors of this study propose that inactivation of APE1 is critical for
the granzyme A-mediated cell death pathway because the transcription
factors activated by APE1 inhibit apoptosis during DNA repair. Others
have suggested that the DNA repair activity of APE1 is responsible for
resistance to mutation-inducing cancer therapies (Fritz et al., 2003).
A similar finding with PARP-1 provides evidence for the role of this
enzyme in regulation of cell fate. PARP-1 is degraded by caspase 3 during
apoptosis (Lazebnik et al., 1994; Nicholson et al., 1995), indicating that it
normally provides resistance to apoptosis. A complementary finding is that
PARP-1 poly(ADP)ribosylates the Ca2þ/Mg2þ endonuclease, suppressing
the action of this proapoptotic enzyme (Yakovlev et al., 2000).
Imbalances in BER are known to induce mutagenesis and to be asso-

ciated with cancer. This phenomenon has been illustrated by overexpres-
sion of DNA glycosylases (Coquerelle et al., 1995; Glassner et al., 1998) or
polymerase (Bergoglio et al., 2002; Canitrot et al., 1998). The increase in
mutagenesis in the former case has been attributed to accumulation
of abasic sites. Overexpression of polymerase is thought to lead to
mutagenesis because of the poor fidelity of this polymerase.
VI. Roles of BER Enzymes in Other Processes
A. BER-Like Enzymes in Plant Development

Accumulating evidence indicates a critical role for proteins with DNA
glycosylase activity in seed development of the plant Arabidopsis thaliana.
The first evidence of this phenomenon came from a study on the imprint-
ing of the MEA gene. This gene is normally silenced by methylation, but
the maternal allele is activated in the endosperm of seeds. The activation
was shown to accompany demethylation of the maternal allele, and the
surprising cause of demethylation was shown to be the DNA glycosylase
DEMETER (Choi et al., 2002). Subsequent studies confirmed this finding
while identifying MET1 as the methyltransferase maintaining MEA in the
repressed state (Xiao et al., 2003) and found that the FWA gene was
regulated in an identical manner, also requiring DEMETER for activation
(Kinoshita et al., 2004). Another Arabidopsis protein related to DEMETER,
ROS1, is a DNA glycosylase that functions in a similar manner. Further-
more, a purified MBP-ROS1 fusion protein was shown to nick (presumably
through bifunctional glycosylase activity) plasmid DNA containing
methylated CpG sites (Gong et al., 2002).
The most intriguing question surrounding this regulatory system is the
exact mechanism whereby the activities of DEMETER and ROS1 lead to
cytosine demethylation at CpG sites. The authors of these studies have
proposed competing mechanisms, invoking either an intrinsic 5-methylcy-
tosine DNA glycosylase activity within DEMETER and ROS1 or an indirect
pathway in which these proteins create single-stranded nicks near methy-
lated CpG sites that somehow lead to replacement with unmethylated
nucleotides. In fact, no mechanism for chromosomal demethylation has
been conclusively established, and reports of 5-methylcytosine glycosylase
activity ( Jost et al., 2001; Zhu et al., 2000) have not been independently
confirmed. It is nonetheless intriguing that DNA glycosylases appear to be
key players in epigenetic demethylation, and we anxiously await further
clarification of the details.
B. Thymine DNA Glycosylase

Thymine DNA glycosylase (TDG) is unique among the DNA glycosylases
with regard to the number and diversity of its protein partners. As
discussed above, the function of TDG is critical for removal of T:G mis-
matches arising via deamination of 5-methylcytosine. TDG has been shown
to be sumoylated at a single-lysine residue (Hardeland et al., 2002).
Sumoylation increases the turnover rate of TDG while suppressing the
activating effect of APE1 by reducing the affinity of TDG for DNA. This
type of regulation is thus far unique to TDG among DNA glycosylase. TDG
is known to interact with the transcriptional coactivators CBP/p300 (Tini
et al., 2002). This interaction stimulates transcription in cells and leads to
acetylation of TDG. The significance of increased transcription may be
that the loosened chromatin structure of a transcriptionally active region
allows DNA repair proteins easier access to the damaged site. Acetylated
TDG no longer binds CBP and no longer recruits APE1 to damaged sites,
indicating that acetylation serves as another level of regulation. TDG has
also recently been reported to function as a coactivator for the nuclear
estrogen receptor alpha (Chen et al., 2003), thus forging yet another link
between BER and other critical aspects of cellular function.
Acknowledgments
We are grateful to Anirban Banerjee for helpful discussions. J.C.F. is sponsored by a Merck-
Wiley Fellowship.
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NUCLEOTIDE EXCISION REPAIR IN E. COLI AND MAN
By AZIZ SANCAR AND JOYCE T. REARDON
Department of Biochemistry and Biophysics,

University of North Carolina School of Medicine,
Chapel Hill, North Carolina, 27599
I. Introduction . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 43
II. Damage Recognition . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 45
III. Mechanism of Excision Repair.. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 48
A. Excision Repair in E. coli . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 49
B. Excision Repair in Humans . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 52
IV. Transcription-Coupled Repair. .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 56
A. Transcription-Coupled Repair in E. coli . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 56
B. Transcription-Coupled Repair in Human Cells .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 57
V. Repair of Chromatin. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 59
VI. Conclusion . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 63
References . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 65
I. INTRODUCTION
Nucleotide excision repair (excision repair) is a universal repair system
that eliminates DNA damage by dual incisions bracketing the lesion.
In nucleotide excision repair, the damage is removed in the form of a
12–13-nucleotide (nt)-long oligomer in prokaryotes and in a 24–32-nt-long
oligomer in eukaryotes (Huang et al., 1992; Sancar and Rupp, 1983)
(Fig. 1) Excision repair comprises three basic steps: damage recognition,
dual incisions and release of the excised oligomer, and resynthesis to fill in
the gap and ligation (Sancar, 1996; Sancar et al., 2004; Wood, 1997).
Nucleotide excision repair is the primary repair system for bulky DNA ad-
ducts such as the cyclobutane pyrimidine dimer (Pyr<>Pyr), (6–4) photo-
product, benzo[a]pyrene-guanine adduct, acetylaminofluorene-guanine
(AAF-G), and cisplatin-d(GpG) diadduct. In addition, it plays a back-up
role for base excision repair by removing nonbulky DNA lesions such as
thymine glycols and 8-oxoguanine at a slow but physiologically relevant
rate. Finally, excision repair is required for interstrand cross-link repair in
Escherichia coli (Van Houten et al., 1986) and participates in one pathway of
cross-link repair in yeast and humans (Bessho et al., 1997; Zheng et al.,
2003).
A defect in nucleotide excision repair causes extreme ultraviolet (UV)
sensitivity in E. coli and Saccharomyces cerevisiae. In humans, defects in

44 SANCAR AND REARDON
FIG. 1. Schematic illustration of nucleotide excision repair in prokaryotes and

eukaryotes. The basic steps are conserved: damage recognition and dual incisions to
excise DNA damage, helicase activity to displace excised oligomer and repair factors, and
resynthesis/ligation to restore the integrity of the DNA molecule. (See Color Insert.)
excision repair cause the inherited disease xeroderma pigmentosum (XP)

(Cleaver, 1968). There are eight XP complementation groups, XP-A
through XP-G and XP-V (variant), and a mutation in any of the genes
can cause the disease. The signs and symptoms of XP include extreme
sensitivity to sunlight, about 10,000-fold increase in skin cancer, and
mental and developmental abnormalities in some cases (Cleaver and
Kraemer, 1989).
Transcription stimulates excision repair both in E. coli (Mellon and
Hanawalt, 1989) and in humans (Bohr et al., 1985; Mellon et al., 1987) in
a process dependent on proteins called transcription-repair coupling fac-
tors (TRCFs). In E. coli the mfd gene encodes the TRCF (Selby and Sancar,
1991, 1993; Selby et al., 1991), and cells mutated in this gene exhibit modest
UV sensitivity but a disproportionately increased rate of UV-induced muta-
tions and lack of mutation frequency decline (mfd) on holding in minimal
medium after irradiation and before plating (Witkin, 1994).
In humans, mutations in the CSA and CSB genes abolish transcription-
coupled repair (Venema et al., 1990a). Cells from Cockayne Syndrome (CS)
patients exhibit phenotypic properties similar to E. coli mfd mutants, which
NUCLEOTIDE EXCISION REPAIR 45
include moderate UV sensitivity and an increased rate of UV mutations that

are mostly caused by lesions in the template strand of transcribed genes
(see Hanawalt, 2002). CS is associated with developmental retardation and
neurological abnormalities but not with an increased incidence of skin
cancer, although the patients are hypersensitive to sunlight. In addition to
mutations in the CSA and CSB genes, some mutations in XPB, XPD, and XPG
also give rise to Cockayne syndrome. In these latter cases, the patients
exhibit a mixed XP/CS phenotype. Because XPB, XPD, and XPG
are involved in transcription in addition to repair and CS is never observed
in the strictly repair-defective XP-A cases, CS is considered a transcrip-
tion defect syndrome rather than a repair deficiency disease. Finally,
a rare genetic disease called trichothiodystrophy (TTD) is also asso-
ciated with mutations in the XPB and XPD genes. TTD patients have scaly,
fishlike skin and brittle hair and nails as a result of a defect in synthesis of
sulfur-rich proteins. TTD patients exhibit some of the developmental and
neurological abnormalities associated with Cockayne syndrome.
In this chapter, we will first address the general problem of damage
recognition specificity by an enzyme system with an essentially infinite
substrate range. Then the mechanisms of DNA repair by the E. coli
and human excision nucleases, as representatives of the prokaryotic and
eukaryotic excision nucleases, will be summarized. The mechanism of
excision repair in S. cerevisiae, which is quite similar to human excision
repair, has been reviewed elsewhere (Prakash and Prakash, 2000) and will
not be discussed here.
II. DAMAGE RECOGNITION

The biological dilemma in DNA repair is the virtually infinite number of
DNA lesions that can and often do form within the lifespan of the cell, the
necessity of repairing these lesions within a lifetime, and the theoretical
limitation on what fraction of the genomic encoding capacity can be
dedicated to repair. It appears that in nature, this problem has been
solved by two main approaches. In one, as in the case of photolyase, a
single enzyme of near-absolute specificity repairs a single, but relatively
abundant, DNA lesion (Sancar, 2003). In the second general scheme,
either a single polypeptide (O’Brien and Ellenberger, 2004) or a multi-
protein enzyme (Sancar et al., 2004) acts on many, often structurally
dissimilar, substrates. The excision nuclease falls into the category of a
multiprotein system with an infinite substrate range, and it must deal with
the physiological necessity of removing all types of lesions, the biological
imperative to avoid normal bases, and the evolutionary requirement of
keeping the mutation load at an optimum.
Three strategies are used by excision nucleases to achieve physiological-

ly acceptable specificity at a biologically relevant rate: cooperativity, mo-
lecular matchmaking, and kinetic proofreading (Fig. 2). In cooperative
DNA–protein interactions, the binding of one protein to DNA facilitates
the binding of either the second subunit of the same protein (homotro-
pic) or of an unrelated protein (heterotropic) by protein–protein interac-
tion. The binding sites of the monomers could be adjacent or overlapping,
and cooperative binding may involve more than two proteins. In qualita-
tive terms, the binding of one protein to DNA facilitates the binding of a
second protein that has a binding site adjacent to the first one by increas-
ing the local protein concentration of the second one by specific protein–
protein interactions. Quantitatively, the binding of the second protein is
enhanced by the first protein by a factor approximately equal to the square
root of the equilibrium association constant of the two proteins (Giedroc
et al., 1987; Kelly et al., 1976). Cooperative DNA binding is important for
all forms of DNA–protein interactions in both prokaryotes and eukaryotes,
FIG. 2. Three mechanisms used to achieve specificity in DNA damage recognition

and repair. (A) Cooperative binding enhances the binding of a second protein to DNA.
(B) Kinetic proofreading achieves specificity by using energy to introduce irreversible
steps (a time delay) between ES and E þ P steps. (C) A molecular matchmaker
promotes the binding of a second protein that is not able to specifically bind DNA on its
own. (See Color Insert.)
and it is the predominant transcriptional regulatory mechanism in

eukaryotes (Levine and Tjian, 2003).
In molecular matchmaking, a protein uses the energy released from
ATP hydrolysis to promote the DNA binding of another protein that is
incapable of binding specifically on its own. A molecular matchmaker is a
protein that brings two compatible yet solitary macromolecules together in
an ATP-dependent reaction, promotes their association, and then leaves
the complex so that the new complex can engage in productive transac-
tions (Orren et al., 1992; Sancar and Hearst, 1993). Because several types
of proteins, including adaptors and mediators, are known to facilitate
DNA–protein interactions, a molecular matchmaker has been defined
narrowly to differentiate it from other facilitators. A protein must fulfill
five criteria to be identified as a matchmaker (Sancar and Hearst, 1993).
First, in the absence of the matchmaker, the affinity of the matched
protein to its target DNA site must be low enough to be physiologically
insignificant. Second, the matchmaker must promote stable complex
formation between DNA and the cognate protein. Third, the matchmaker
or the cognate protein must hydrolyze ATP to generate the energy needed
for complex formation. Fourth, the matchmaker must make a ternary
complex with the matched DNA and protein, causing a conformational
change but no covalent modification. Fifth, after stable complex forma-
tion, the matchmaker must dissociate to allow the matched protein to
carry out its effector function. In nucleotide excision repair, UvrA in E. coli
and XPC in humans fulfill all five criteria of molecular matchmakers.
Molecular matchmaking provides a stepwise recognition mechanism and
ATP hydrolysis-dependent exposure of the interacting groups on DNA
and protein to achieve specificity. However, neither cooperativity nor
molecular matchmaking are sufficient to confer the specificity and rate
requisite for a biological system. Thus, a third mechanism, kinetic proof-
reading, complements these thermodynamic molecular mechanisms to
achieve the necessary specificity.
Kinetic proofreading is the biochemical version of information theory,
whereby energy is converted into information. Kinetic proofreading is a
specificity mechanism that achieves high fidelity beyond the level that can
be achieved by the free-energy difference between correct and incorrect
interactions, through the presence of (unidirectional) energy-using inter-
mediate steps at each of which the reaction can be aborted (Hopfield,
1974; Ninio, 1975). This specificity mechanism is equivalent to the intro-
duction of a time delay between ES complex formation and the formation
of product in an otherwise normal Michaelis–Menton scheme.
When applied to specific biological systems, there are variations on
the original scheme (Burgess and Guthrie, 1993; MacGlashan, 2001).
However, for a particular cellular response to occur, kinetic proofreading

in general requires that the enzyme–substrate (or DNA–protein) complex
complete a series of irreversible reactions (such as ATP hydrolysis or
protein phosphorylation) while a moderate-specifcity ligand is bound; this
temporally separates the binding step from the effector step (in nucleotide
excision repair, the dual incision event). If the ligand dissociates before
the full set of either covalent modifications or conformational changes is
completed, the reaction is aborted and must restart at the beginning.
Thus, the kinetic proofreading mechanism differs from other multiple-
step kinetic reaction schemes of the Michaelis–Menton type in which every
step but the ultimate one (EP ! E þ P) is reversible. As an illustration of
the power of kinetic proofreading in conferring specificity, a 10-fold
difference between the off rates (e.g., 0.1 s1 and 1.0 s1) between specific
and nonspecific complexes can provide 104-fold difference in the effec-
tor reaction by interposing five steps between binding and catalysis steps.
In nucleotide excision repair, UvrA and UvrB in E. coli and XPB and XPD
in humans are ATPases that hydrolyze ATP to create irreversible inter-
mediates between the initial low-specificity binding and the final dual-
incision steps and, thus, perform kinetic proofreading. It must be obvious,
however, from this brief description of kinetic proofreading that the
specificity conferred by this mechanism is not absolute. Actually, some
degree of nonspecific effector reaction is intrinsic to the kinetic proof-
reading scheme, and the production of side products is a hallmark of a
reaction pathway that employs kinetic proofreading. In the case of nucle-
otide excision repair, the kinetic proofreading scheme would predict
excision of undamaged bases. Indeed, both in E. coli and in humans,
excision repair acts on undamaged DNA excising damage-free oligomers
12–13 nt and 24–32 nt in length, respectively (Branum et al., 2001).
III. MECHANISM OF EXCISION REPAIR

In E. coli, dual incisions are accomplished by three proteins (UvrA,
UvrB, and UvrC), and in humans 15 polypeptides in six repair factors
carry out the same task. The properties of the E. coli and human excision
repair factors are summarized in Tables I and II, respectively. Of signifi-
cance, in contrast to all other repair systems, the prokaryotic and eukary-
otic excision repair factors are evolutionarily not related and show
no sequence homology to one another. However, the basic strategies for
the prokaryotic and eukaryotic excision nucleases are similar. First, dam-
age is recognized by an ATP-independent mechanism to form an unsta-
ble DNA–protein complex. Then this complex is converted to a stable
preincision form by ATPase subunits that hydrolyze ATP and unwind the
TABLE I
Subunits of the Escherichia coli Excision Nuclease
Protein Mr Motif/Domain Function
UvrA (104)2 ABC superfamily Damage recognition

2 zinc fingers Molecular matchmaker
UvrB 78 Helicase motif Damage recognition
DNA unwinding
Contributes to 30 incision
UvrC 69 GIY-YIG nuclease GIY-YIG: 30 incision
EndoV nuclease EndoV: 50 incision
TABLE II
Subunits of the Human Excision Nuclease
Factor Proteins Activity Role in repair
XPA XPA/p31 DNA binding Damage recognition

RPA p70 DNA binding Damage recognition
p32 Replication factor
p14
XPC p106 DNA binding Damage recognition
HR23B/p58 Molecular matchmaker
TFIIH XPB/ERCC3/p89 DNA-dependent DNA unwinding
XPD/ERCC2/p80 ATPase Kinetic proofreading
p62 Helicase
p52 General transcription
p44 factor
p34
XPG XPG/ERCC5/p135 Nuclease 30 incision
XPFERCC1 XPF/ERCC4/p112 Nuclease 50 incision
ERCC1/p33
duplex to promote the formation of more intimate protein–DNA contacts.

Finally, the dual incisions are made in a concerted, but asynchronous,
manner such that the 30 incision precedes the 50 incision.
A. Excision Repair in E. coli

In E. coli, the dual-incision activity is carried out by the sequential and
partially overlapping functions of UvrA, UvrB, and UvrC (Fig. 3), and
the activity is referred to as (A)BC excision nuclease or (A)BC excinu-
clease (Orren and Sancar, 1989; Sancar and Rupp, 1983). The name is
descriptive of the reaction performed by the enzyme system (excising an
FIG. 3. Model for excision repair in E. coli. UvrA dimerizes (cooperativity) and
interacts with UvrB to form an A2B1 heterotrimer, the damage recognition factor. UvrA
delivers UvrB to the damage site and then dissociates (molecular matchmaker). UvrC
recognizes and binds to the UvrB–DNA complex, in which the DNA is bent and locally
unwound. ATP hydrolysis introduces irreversible intermediates at steps along the
pathway leading to dual incision, and the reaction may be aborted at any step (kinetic
proofreading). Dual incisions release the damage in a 12–13-nt-long oligomer. UvrD
(Helicase II) displaces UvrC and the excised oligomer, and then DNA polymerase I
displaces UvrB during resynthesis to fill in the gap; newly synthesized DNA is ligated to
complete the repair reaction. (See Color Insert.)
oligomer from DNA) and is preferable over other names such as UvrABC
endonuclease or UvrABC nuclease, which give the connotation of nicking
or degrading the DNA.
In an ATP-dependent reaction, UvrA and UvrB make an A2B1 hetero-
trimer that is the damage recognition complex (Orren and Sancar, 1989).
The damaged DNA preference of this complex is conferred by UvrA, which
even on its own is capable of preferentially binding to bulky lesions such as
a psoralen-thymine monoadduct (Van Houten et al., 1987) and acetylami-
nofluorene-guanine adduct (Bertrand-Burggraf et al., 1991; Delagoutte
et al., 1997, 2002). Thus, the A2B1 complex, guided by the preference of
UvrA for damaged DNA, binds at the damage site and the ‘‘helicase’’
activity of UvrB unwinds DNA at this site by about 5 bp and kinks it by 130
degrees. This kinking and unwinding is accompanied by significant con-
formational changes in UvrB as well (Delagoutte et al., 2002; Goosen et al.,
1998; Hsu et al., 1995; Shi et al., 1992; Zou and Van Houten, 1999). These
changes lead to formation of a stable UvrB–DNA complex and dissociation
of (UvrA)2 from the DNA (Orren and Sancar, 1989; 1990). Once UvrA
dissociates, UvrC binds with high affinity and specificity to the B1-DNA
complex, and the dual incisions are made (Orren and Sancar, 1989; Orren
et al., 1992). The 30 incision is made by the GIY-YIG homing endonuclease-
related N-terminal domain of UvrC (Aravind et al., 1999; Kowalski et al.,
1999; Verhoeven et al., 2000) with participation of some residues from
UvrB (Lin and Sancar, 1991; Lin et al., 1992). This is rapidly followed by
the 50 incision made by the catalytic ‘‘triad’’ comprising Asp399, Asp438,
Asp466, and His538 in the C-terminal EndoV domain of UvrC (Lin and
Sancar, 1992). The 30 incision is made at the fourth through the sixth
phosphodiester bond, and the 50 incision is made at the eighth phospho-
diester bond. Thus, regardless of the type of damage, the lesion is excised
in the form of a 12–13-nt-long oligomer.
Interestingly, E. coli and some other bacteria contain a protein that is
homologous to the N-terminal half of UvrC (Moolenaar et al., 2002). This
protein is called Cho (UvrC homologue) and, acting in conjunction with
UvrA and UvrB, makes the 30 incision at the ninth phosphodiester bond 30
to the lesion but cannot make the 50 incision. The deletion of cho has no
measurable effect on UV survival of wild-type E. coli and only a minor effect
on E. coli lacking the UvrC protein. It appears that Cho, working coordi-
nately with UvrC, makes some contribution to DNA repair by the (A)BC
excinuclease.
Following the dual incisions, UvrB, UvrC, and the ‘‘excised’’ oligomer
remain in the postincision complex, although the excised oligomer is no
longer hydrogen-bonded to the complementary strand (Orren et al., 1992).
UvrC is not very stably bound in this complex, and its dissociation is
accelerated by UvrD (helicase II), which also releases the excised oligomer.
UvrB remains bound in the excision gap and is displaced by DNA polymer-
ase I concomitant with gap filling to produce a repair patch exactly match-
ing the size of the excised oligomer (Sibghat-Ullah et al., 1990). Finally, the
newly synthesized DNA is ligated to complete the repair reaction.
As noted above, the (A)BC excinuclease has a virtually infinite substrate
range; however, it is most effective on lesions such as (6–4) photoproducts
and AAF–guanine adducts that destabilize the duplex, as well as, interest-
ingly, on the thymine–psoralen monoadduct that stabilizes the helix (see
Petit and Sancar, 1999). The enzyme system uses cooperativity, molecular
matchmaking, and kinetic proofreading to achieve specificity. The coop-
erative interactions include facilitation of UvrA binding to DNA by photo-
lyase (Sancar et al., 1984) and the dimerization of UvrA on DNA. UvrA is
the molecular matchmaker that delivers UvrB to DNA, and UvrA must
dissociate before UvrC can bind to UvrB–DNA and make the dual inci-
sions. Thus, UvrA contributes to specificity both in selective loading of
UvrB and in dissociation from the UvrB–DNA complex. Kinetic proof-
reading encompasses the unwinding of DNA by the A2B1 complex, hydro-
lysis of ATP by UvrB in the UvrB–DNA complex (Delagoutte et al., 2002),
and differential affinities of UvrC to various UvrB–DNA complexes. No
quantitative estimates are currently available for the contributions of the
various mechanisms to specificity. However, it is likely that kinetic proof-
reading is the predominant determinant of specificity because the ther-
modynamic discrimination by UvrA (or A2B1) between undamaged and
damaged DNA is almost nonexistent for such important lesions as cyclo-
butane pyrimidine dimers (Bertrand-Burggraf et al., 1991) that, in the
absence of light or photolyase, can be repaired only by (A)BC excinu-
clease. As indicated above, kinetic proofreading is a powerful mechanism
for achieving specificity, but by its very own design it has a built-in error
rate such that nonsubstrates are also processed at significant rates. This is
true for (A)BC excinuclease as well. The enzyme attacks undamaged DNA
at about 10-4 the rate of cyclobutane pyrimidine dimer, excising damage-
free 12–13-nt-long oligomers. This excision results in ‘‘gratuitous repair’’
(Branum et al., 2001), which may be stimulated by specific DNA structures
or DNA dynamics such as transcription (Hanawalt, 2002), and that might
be a source of spontaneous mutagenesis.
B. Excision Repair in Humans

Excision repair in humans is carried out by six repair factors: RPA, XPA,
XPC, TFIIH, XPG, and XPFERCC1 (Table II). RPA, XPA, and XPC
recognize the damage; TFIIH unwinds the duplex around the damage;
and XPG and XPFERCC1 make the 30 and 50 incisions, respectively (Evans
et al., 1997; Mu et al., 1995, 1996). Of the three proteins known to
recognize DNA damage (RPA, XPA, and XPC), there has been serious
debate as to which binds damage first and therefore should be called the
‘‘damage sensor.’’ Using either AAF–guanine adducts or (6–4) photopro-
ducts as substrates for repair assays, it has been variously concluded that
XPC or RPA and XPA are the sensors (Missura et al., 2001; Sugasawa et al.,
1998; Wakasugi and Sancar, 1998, 1999). Although the causes of the
discrepancies among the various groups remain to be investigated, it is
important to note that all groups working on human excision repair agree
that XPC does not recognize cyclobutane pyrimidine dimers (Batty et al.,
2000; Reardon and Sancar, 2003; Sugasawa et al., 2001). In fact, there is a
report indicating that XPC prefers undamaged DNA over cyclobutane
pyrimidine dimer-containing DNA, and thus it appears XPC avoids this
lesion (Hey et al., 2002). There seems to be a consensus that the most
important substrate for the human excision nuclease is not recognized
by XPC, and therefore, in the case of Pyr<>Pyr repair, XPC cannot be
the initiator. However, XPA and RPA are equally inefficient in recogniz-
ing Pyr<>Pyr, and by this criterion, they cannot be the initiators either
(Reardon and Sancar, 2003). The damaged DNA-binding protein (DDB)
is a p127p48 heterodimer, and the small subunit of DDB is encoded by
the XPE gene (Nichols et al., 1996). It has been reported that DDB binds to
Pyr<>Pyr with modest affinity (Wakasugi et al., 2001; 2002), and hence it
might be the ‘‘initiator’’ for repair of Pyr<>Pyr. However, this report is at
odds with findings that CHO cell extracts, which do not contain DDB
because of promoter silencing of the p48(DDB2) gene, are quite efficient
at excising Pyr<>Pyr (Reardon et al., 1997a) and that supplementing the
extracts with DDB has no effect on repair at low concentrations and
inhibits repair at high concentrations (Reardon and Sancar, 2003).
With this background, the following scheme has been proposed for the
initial assembly at Pyr<>Pyr and at all other lesions: RPA, XPA, and XPC
[usually in the form of XPCTFIIH (Drapkin et al., 1994)] have some
preference for all DNA lesions, and any of the three may bind first. These
proteins also have affinities to one another: XPA binds to both RPA and
the XPCTFIIH complex (Li et al., 1995; Park et al., 1995). Thus, the three
damage recognition components act cooperatively and assemble in ran-
dom order at damage sites. Cooperative interaction increases the specific-
ity somewhat, but the discrimination between damaged and undamaged
DNA by each of the components and the affinities of the three factors to
one another are of insufficient magnitude to confer a physiologically
relevant specificity. A model consistent with all existing data, incorporat-
ing cooperativity, molecular matchmaking, and kinetic proofreading, has
FIG. 4. Model for excision repair in man. The damage recognition factors,
RPA, XPA, and XPCTFIIH, assemble at the damage site in a random order but in a
cooperative manner to form an unstable ‘‘closed’’ complex. ATP hydrolysis by
been proposed for human excision nuclease (Reardon and Sancar, 2003,
2004; Wakasugi and Sancar, 1998) and is as follows (Fig. 4).
The damage recognition factors, RPA, XPA, and XPCTFIIH, assemble at
the damage site in a random order but in a cooperative manner and form
an unstable ‘‘closed complex.’’ The moderate specificity achieved by coop-
erative binding is amplified by the kinetic proofreading activity of TFIIH,
a six-subunit transcription/repair factor with both 30 ! 50 and 50 ! 30
helicase activity (Egly, 2001). Two subunits of TFIIH, XPB and XPD,
hydrolyze ATP and unwind the duplex at the damage site to form a repair
bubble of about 20 nt (Evans et al., 1997; Mu et al., 1997). This unwinding
is accompanied by significant conformational changes in all components
of the complex, leading to a new set of interactions that produces a rather
stable complex called preincision complex 1 (PIC1). XPC is a molecular
matchmaker that uses the ATP hydrolysis activity of TFIIH to promote
entry of XPG into the complex as XPC leaves. The resulting complex is
called PIC2. Finally, XPFERCC1 binds PIC2 to form PIC3 in which
XPG makes the 30 incision first, followed by the 50 incision made by
XPFERCC1. The first incision is made at the sixth 3 phosphodiester
bond 30 to the damage and the second incision is made at the twentieth 5
phosphodiester bond 50 to the lesion to generate a damage-containing
oligomer of 24–32 nt (Huang et al., 1992). The excised oligomer and most
of the repair factors dissociate from the duplex (Mu et al., 1996, 1997),
leaving RPA in the gap. Then repair synthesis proteins RFC/PCNA and Pol
/" fill in the gap, and the repair patch is sealed by DNA ligase 1. As in the
case with E. coli excision repair, the repair patch exactly matches the size of
the excision gap (Reardon et al., 1997a).
The human excision nuclease has an essentially infinite substrate range
(Branum et al., 2001; Huang et al., 1994; Reardon et al., 1997b), and as in
the case with E. coli, the excision nuclease employs cooperativity, molecu-
lar matchmaking, and kinetic proofreading to remove damage while
minimizing the attack on undamaged DNA. Because of the larger ge-
nome size, human cells make more extensive use of both cooperativity
and kinetic proofreading in DNA repair. Damage recognition involves
TFIIH unwinds the duplex around the lesion, causing formation of a stable complex
called preincision complex 1 (PIC1). XPC is a molecular matchmaker that helps to
recruit/deliver XPG to PIC1, and XPC leaves before formation of PIC2, which
comprises XPA, RPA, TFIIH, and XPG. Finally, this complex is recognized by
XPFERCC1, leading to formation of PIC3 and the dual incision event, which releases
damage in a 24–32-nt-long oligomer. ATP hydrolysis is required for PIC1–PIC3
formation, and the reaction may be aborted at any step along the pathway (kinetic
proofreading) leading to dual incision. The repair gap is filled in by polymerase /",
with the aid of RFC/PCNA, and sealed by DNA ligase. (See Color Insert.)
cooperative interactions among RPA, XPA, and XPCTFIIH, and there are
at least three proofreading steps at the formation of PIC1, PIC2, and PIC3,
where ATP hydrolysis creates intermediates that cannot revert to the
previous step but that may revert to the preassembly step at each of the
stages if assembly occurred at a site with no DNA damage. At present, we
do not have quantitative data on the relative contributions of coopera-
tivity and kinetic proofreading to the specificity of excision repair in
human cells. As in the case with the E. coli excinuclease, the human
excision nuclease attacks and excises undamaged DNA at a significant
rate (Branum et al., 2001; Reardon and Sancar, 2003). Clearly, the
biological necessity of repairing all DNA lesions within the confines of
the cellular limitation on the number of enzymes comes at a cost, which is
a low frequency of mutations that inevitably occur when the gaps formed
by excising undamaged DNA are filled in by DNA polymerases.
IV. TRANSCRIPTION-COUPLED REPAIR

Excision repair is affected by other DNA transactions, including binding
of regulatory proteins, compaction into chromatin, replication, recombi-
nation, and transcription. It has been found that transcription stimulates
excision repair both in E. coli and in humans (Bohr et al., 1985; Mellon and
Hanawalt, 1989; Mellon et al., 1987). Moreover, in the majority of cases,
transcription stimulates the repair of only the transcribed strand (Mellon
et al., 1987), and it may actually inhibit repair of the transcribed strand in
the absence of an active mechanism coupling the two processes (Selby
and Sancar, 1990). In the case of E. coli, the mechanism of transcription-
coupled repair is reasonably well understood. In contrast, there is no
in vitro system for eukaryotic transcription-coupled repair, and hence
the mechanistic aspects of this process remain to be elucidated.
A. Transcription-Coupled Repair in E. coli

Transcription-coupled repair is responsible for reduction of the muta-
tion frequency (mutation frequency decline) in E. coli cells that are held in
minimal medium after UV irradiation before plating on a rich selection
medium (Li et al., 1999; Witkin, 1994). Transcription is coupled to exci-
sion repair through the intermediacy of the ‘‘transcription-repair coupling
factor’’ (TRCF) encoded by the mfd gene (Selby et al., 1991). TRCF is a
130-kDa monomer, possesses helicase motifs, and functions as a translo-
case on RNA polymerase, causing its progression at temporary pause sites
but releasing it when elongation is blocked by DNA damage or by a tightly
bound protein (Park et al., 2002; Selby and Sancar, 1995; Washburn et al.,
2003). Transcription-coupled repair in E. coli proceeds as follows (Selby
and Sancar, 1993) (Fig. 5): E. coli RNA polymerase is unaffected by a DNA
lesion such as Pyr<>Pyr in the nontranscribed strand, but when the
damage is in the transcribed strand, elongation is blocked. The ternary
complex that forms at a damage site is very stable with a half-life of greater
than 20 hours and inhibits excision repair by interfering with the binding
of the A2B1 complex. The TRCF recognizes both the stalled RNA poly-
merase and UvrA in the A2B1 complex. It releases RNA polymerase and
the truncated transcript while simultaneously recruiting the A2B1 complex
to the damage site. After the delivery of A2B1 to the lesion, TRCF dis-
sociates, enabling UvrA to load UvrB onto the lesion, followed by binding
of UvrC and excision of the damage.
Because damage recognition is presumed to be the rate-limiting step in
excision repair, and because a stalled RNA polymerase is a high-affinity
target for TRCF, the overall effect of the process is an increase in the rate
of repair of the transcribed strand relative to the coding (nontranscribed)
strand and nontranscribed DNA. Thus, in transcription-coupled repair,
RNA polymerase plays the role of a damage recognition subunit of the
excision nuclease. This phenomenon has been called ‘‘recognition by
proxy’’ (Sancar et al., 2004). In mfd mutants, repair of the transcribed
strand is inhibited by the stalled RNA polymerase and, as a consequence,
the coding strand and nontranscribed DNA are repaired more efficiently
than the template strand of transcribed genes.
B. Transcription-Coupled Repair in Human Cells

Transcription-coupled repair occurring at genes transcribed by RNA
polymerase II (RNAPII) requires both excision repair factors and the
CSA and CSB proteins (see Hanawalt, 2002; Venema et al., 1990a). Inter-
estingly, XPC is not needed for this process (Venema et al., 1990b). The
CSA protein belongs to the WD40 family of proteins (Henning et al.,
1995), but how CSA may function in coupling transcription to repair is
not clear. In contrast, the CSB protein, like the E. coli TRCF, possesses
helicase motifs (Troelstra et al., 1992) and has some properties analogous
to E. coli TRCF: it is an ATPase but not a helicase, and it has a translocase
activity that enables RNAPII to progress through natural transcription
pause sites (Selby and Sancar, 1997a,b). However, in contrast to the
E. coli TRCF, the CSB protein does not disrupt the ternary complex of
stalled RNA polymerase (Selby and Sancar, 1997b). At present, there is no
in vitro system in which transcription by RNAPII stimulates excision repair.
FIG. 5. Model for transcription-coupled repair in E. coli. A lesion in the

nontranscribed (NT) strand has no effect on RNA polymerase (RNAP) (left side),
but a lesion in the transcribed strand blocks progression of RNAP (right).
Transcription-repair coupling factor (TRCF) recognizes and binds to the stalled
However, three observations are relevant to how transcription may

stimulate repair. First, a bubble structure 30 to a Pyr<>Pyr, such as might
occur during transcription, is a substrate for human excision nuclease
without XPC (Mu and Sancar, 1997). Second, surprisingly, RNAPII stalled
at a Pyr<>Pyr does not inhibit excision of the lesion by the reconstituted
human excision nuclease (Selby et al., 1997). Third, the human transcrip-
tion termination factor 2 (TTF2) releases RNAPI and RNAPII stalled at
a lesion and, in this regard, functions like the E. coli TRCF (Hara et al.,
1999).
Taking these facts into account, the following are two plausible models
for transcription-coupled repair in humans (Fig. 6). The DNA in human
cells is packed into chromatin, a structural feature that inhibits excision
repair (see following). Transcribed genes are characterized by an open
chromatin conformation, and stalling of RNAPII at a lesion may preserve
this open structure, thus targeting the transcription-blocking lesion for
rapid repair and simultaneously avoiding the inhibitory effect of chroma-
tin compaction. Although CSB does not disrupt the ternary complex, it
does interact with essential repair factors (Iyer et al., 1996; Selby and
Sancar, 1997a), and thus an active role in transcription-coupled repair
cannot be ruled out. It is possible that TTF2, which releases RNAPI and
RNAPII stalled at a lesion (Hara et al., 1999), is the human TRCF but there
is no evidence that TTF2 interacts with and recruits repair factors to sites
of DNA damage. Clearly, more research is required to distinguish among
these possibilities and determine the molecular mechanism of transcrip-
tion-coupled repair in human cells, including the biochemical functions
of CSA, CSB and TTF2 in the process.
V. REPAIR OF CHROMATIN
Eukaryotic chromosomes are packaged into chromatin, a compact
structure made up, at the first level of compaction, of DNA tightly wrap-
ped around a histone octamer (nucleosome) that is joined to neighbor-
ing nucleosomes through linker DNA associated with a linker histone
(Kornberg and Lorch, 1999; Wolffe, 1997). This structural organization
has a significant influence on the distribution of UV-induced damage
within chromatin (nucleosome vs. linker) and within the nucleosome core
polymerase and also binds to UvrA in the A2B1 complex. In an ATP-dependent

reaction, TRCF displaces RNAP and the truncated transcript and, as RNAP
leaves, UvrA2B1 replaces it at the damage site. UvrA delivers UvrB, UvrC binds to the
UvrB–DNA complex, and excision proceeds as illustrated in Fig. 3. (See Color Insert.)
FIG. 6. Model for transcription-coupled repair in human cells. Transcribing RNA

polymerase (RNAP) is blocked by DNA lesions. Two scenarios are plausible and
consistent with the available data. To the left is a pathway in which repair factors (XPC is
not required) assemble at the lesion site and excise the damage, unaffected by the
presence of the stalled polymerase and its transcript. After repair, RNA polymerase
continues to translocate and transcribe. The pathway illustrated on the right involves
transcription termination factor 2 (TTF2), which in an ATP-dependent reaction,
displaces the stalled polymerase; TTF2 leaves the DNA, having performed its function.
Repair is initiated and proceeds as illustrated in Fig. 4. Mechanistic details for the
involvement of CSA and CSB are not known, and thus, they are shown ambiguously
between the two pathways. (See Color Insert.)
(Gale and Smerdon, 1990; Gale et al., 1987; Mitchell et al., 1990; see
Smerdon, 1991). In addition to this effect on adduct distribution, packag-
ing of DNA into nucleosomes represses various DNA transactions by
interfering with the accessibility of DNA-processing enzymes, including
repair factors (Meijer and Smerdon, 1999; Moggs and Almouzni, 1999;
Thoma, 1999).
Indeed, packing DNA into minichromosomes results in less efficient
repair than that observed in naked DNA, presumably because of reduced
accessibility of the repair factors (Sugasawa et al., 1993; Wang et al., 1991).
These studies used randomly damaged DNA and could not distinguish
between inhibition of repair in the nucleosome core or linker DNA. More
recent studies have used the in vitro assembly of nucleosomes containing
site-specific DNA lesions within the core particle and either purified repair
factors or mammalian cell extracts to examine in more detail the effect
of chromatin structure on excision repair. It was determined that UV-
induced photolesions as well as AAF- and cisplatin-modified bases located
in nucleosome cores are repaired at a 5- to 10-fold reduced rate relative to
the same lesions within the same sequence context in naked DNA (Hara
and Sancar, 2002, 2003; Hara et al., 2000; Wang et al., 2003).
These results are consistent with an effect of chromatin on protein
accessibility, a problem that has been extensively studied with respect to
transcription (Workman and Kingston, 1998). There are two major classes
of chromatin-modifying factors that increase the accessibility of transcrip-
tion factors to DNA in chromatin and thus, by analogy, may enhance
the accessibility of repair factors to DNA damage within nucleosome cores
(Aalfs and Kingston, 2000). The first class alters DNA-histone interactions
through covalent modification of histones (Strahl and Allis, 2000). The
second class encompasses several multisubunit ATP-dependent chroma-
tin-remodeling complexes, including SWI/SNF2, ISWI, and Mi-like
complexes. In one study, it was determined that ACF (ISWI-like)
stimulated excision of (6–4) photoproducts in the linker region but had
no effect on repair in the nucleosome core (Ura et al., 2001). In contrast,
SWI/SNF (Kassabov et al., 2003; Yudkovsky et al., 1999) stimulated the
repair of AAF-G adducts and (6–4) photoproducts, but not cyclobutane
pyrimidine dimers, located in the nucleosome core (Hara and Sancar,
2002, 2003). It was found that the three damage recognition factors, RPA,
XPA, and XPC, stimulate the remodeling activity of SWI/SNF, which in
turn enhances excision of DNA lesions in the nucleosome core. The data
indicate a plausible model for the role of SWI/SNF in excision repair
(Hara and Sancar, 2002) (Fig. 7): repair factors locate the damage and
facilitate recruitment of SWI/SNF, which remodels the nucleosome and
facilitates the entry of XPG and XPFERCC1, leading to dual incisions and
release of the damage-containing oligomer. Alternatively, remodeling by
SWI/SNF may facilitate the assembly of repair factors at the damage site.
More work is needed to distinguish between the two possibilities. Repair of
chromatin is a relatively new and unexplored aspect of DNA repair in
human cells, and future research will provide insight into the participation
of various chromatin-modifying factors on repair in nucleosomes and
in higher orders of DNA compaction.
FIG. 7. Model for the role of chromatin remodeling by SWI/SNF in excision repair.
Two pathways are presented: repair factors first or SWI/SNF first. To the left is a pathway
in which damage recognition factors assemble at the damage site and then recruit SWI/
SNF to remodel the nucleosome. To the right is an alternative pathway in which
remodeling by SWI/SNF accelerates the assembly of repair factors at the damage site. In
both cases, dual incisions require the full complement of repair factors, as illustrated in
Fig. 4, and after repair synthesis, the nucleosomes are reassembled. (See Color Insert.)
VI. CONCLUSION
Nucleotide excision repair is the major cellular pathway for removal
of bulky lesions such as those introduced by UV irradiation or chemical
carcinogens. Failure to remove DNA damage can result in increased
mutagenesis, cancer, and cell death. Compared to the prokaryotic excision
nuclease, the eukaryotic system is more complex, requiring 15 polypep-
tides for the basal reaction (recognition and removal of damage in naked
DNA), a process that is accomplished by three proteins in bacteria.
Although the human excision nuclease components show no homology
to the prokaryotic proteins, the overall strategy is the same: damage
recognition, localized helix unwinding, dual incisions to remove the
lesion, and resynthesis/ligation to restore the DNA molecule.
The mechanistic details of the dual-incision event are well characterized,
especially in bacteria, so it was quite surprising when Goosen and collea-
gues reported the discovery of Cho, a previously unknown UvrC homolog
that functions in E. coli excision repair (see Van Houten et al., 2002). Are
there other repair protein homologs, particularly in the more complex
human cell, and what role might they have in excision repair?
Both prokaryotic and eukaryotic excision nucleases recognize and
repair a wide spectrum of lesions, albeit with different efficiencies. Pre-
cisely how a DNA-binding protein distinguishes between normal and
abnormal bases (base pairs) is not known. Thermodynamic destabilization
by damage is a commonly proposed mechanism (see Geacintov et al.,
2002), but such a mechanism disregards helix-stabilizing lesions such
as those introduced by psoralen that are repaired efficiently (see Isaacs
and Spielmann, 2004). Continued investigations are necessary to charac-
terize the structural features that permit damage recognition factors to
discriminate damaged from nondamaged DNA.
Aside from this very basic question of what structural features make
an adducted base abnormal, the more general question of damage recog-
nition in human cells remains unresolved. Although there is a single
damage recognition factor in E. coli (the A2B1 heterotrimer), human
cells have three essential repair factors that show some affinity for various
types of DNA damage: RPA, XPA, and XPC. In contrast to previous
models that assigned a specific protein the role of ‘‘initiator,’’ we recently
suggested that the three damage recognition factors assemble randomly
at sites of DNA damage (Reardon and Sancar, 2003). Furthermore, we
proposed a model in which this random assembly is accompanied
by cooperative DNA binding, molecular matchmaking, and kinetic
proofreading to achieve the requisite specificity in damage recognition
and repair. This is a model to be tested, and further research is needed
to ascertain the relative contributions of cooperativity and kinetic

proofreading to the specificity of excision repair in human, as well as
bacterial, cells.
Excision repair is modulated by transcription in both eukaryotic and
prokaryotic cells. The phenomenon of transcription-coupled repair
(TCR) was first identified in mammalian cells, but in contrast to the
well-established mechanistic details of TCR in E. coli, we have only a
rudimentary understanding of the analogous system in human cells.
Elucidation of the mechanistic aspects of this process first requires the
development of an in vitro system in which both transcription and excision
repair are accomplished at the efficient levels necessary for detailed
biochemical studies. The problem of repair in chromatin is unique to
eukaryotic cells and is likely relevant to the issue of transcription-coupled
repair in human cells. Although recent work has provided insight into
how lesions located in inaccessible regions of chromatin are repaired,
continued research will reveal new details of this intriguing aspect of
DNA repair. Eukaryotic cells have a complex system of checkpoints that
delay or arrest cell cycle progression in response to DNA damage (see
Sancar et al., 2004). How DNA repair is integrated into this response is an
area for future study.
With the exception of the XP-E complementation group, the gene
products of all XP genes have well-defined roles in either excision repair
or translesion synthesis (XP-V). The XPE gene encodes the small subunit
of DDB, an abundant damaged DNA-binding protein. Much is known
about DDB (see Tang and Chu, 2002), but its role in the cellular response
to DNA damage has not been resolved and remains an active area of
research. Among human proteins, DDB has the best discriminatory power
between damaged and undamaged DNA, but it does not have a major role
in excision repair, as evidenced by near-normal levels of excision and
repair synthesis in vivo and by the in vitro reconstitution of excision repair
without DDB (see Reardon and Sancar, 2003). How, then, is DDB involved
in the cellular response to DNA damage? It has been suggested that DDB
functions in the repair of lesions in chromatin as part of a multiprotein
complex that performs chromatin remodeling in vivo (see Wittschieben
and Wood, 2003). Although DDB does not stimulate repair of nucleoso-
mal DNA in vitro (Hara et al., 2000), it possibly functions in this capacity in
vivo, where there are higher orders of DNA compaction. DDB seems to be
involved in other cellular processes, including transcription and the repli-
cation checkpoint, and it may function as a tumor suppressor by regula-
tion of the p53 protein that controls cell-cycle progression and apoptosis
following DNA damage (Itoh et al., 2003; see Hanawalt, 2002). Clearly,
much work is needed to determine the in vivo functions of DDB. Such
studies will also provide additional insight into the mechanisms of DNA
repair in human cells and how this very important enzymatic pathway
contributes to the avoidance of cancer.
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PHOTOLYASE AND CRYPTOCHROME
BLUE-LIGHT PHOTORECEPTORS
By AZIZ SANCAR

University of North Carolina,
Chapel Hill, North Carolina, 27599
I.Introduction . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 73
II.Phylogenetics . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 74
III.Structure of Photolyase. . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 77
IV. Reaction Mechanism of Photolyase . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 79
A. Binding of Photolyase to Substrate. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 81
B. Catalysis by Photolyase. . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 83
V. (6–4) Photolyase. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 86
A. Binding of (6–4) Photolyase. . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 87
B. Catalysis by (6–4) Photolyase . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 88
VI. Cryptochrome . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 90
A. Structure. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 91
B. Function of Cryptochrome . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 92
VII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 96
References . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 96
I. Introduction
Photolyase repairs ultraviolet (UV)-induced DNA damage using near-
UV/blue-light as energy source or cosubstrate (Fig. 1). The enzyme is a
monomeric protein that contains two chromophore/cofactors (Sancar,
1994, 2003). One of the chromophores, which in the majority of
photolyases is methenyltetrahydrofolate (MTHF) and in a limited number
of species that can synthesize 5-deazaflavin is 8-hydroxy-7,8-didmethyl-5-
deazariboflavin (8-HDF) (Fig. 2), is located on the surface of the protein
and functions as a photoantenna. The catalytic cofactor, located in the
core of the globular enzyme, is the two-electron reduced and
deprotonated flavin, FADH. Cryptochrome is defined as a photolyase-
like molecule with no DNA repair activity (Cashmore, 2003; Sancar, 2000,
2003). Cryptochromes regulate growth and development in response to
blue-light in plants and control the circadian clock in animals by light-
dependent and light-independent mechanisms. The function of
cryptochromes in bacteria is not known at present.

74 SANCAR
Fig. 1. (A) Photoreactivation. Escherichia coli cells exposed to the indicated doses of
254-nm ultraviolet (UV) were either plated directly (closed circles) or exposed to a
white-light flash of about 1 ms before plating (open circle). Following incubation at

37 C for 24 hours, colonies were counted and relative survival was calculated. (B)
Molecular basis of UV killing and photoreactivation. Far UV (200–280 nm) induces two
major photoproducts in DNA: the cyclobutane pyrimidine dimer (Pyr<>Pyr) and the
pyrimidine–pyrimidone (6–4) photoproduct. Thymine is the most common pyrimidine
in both types of photoproducts. These photoproducts are reversed to normal bases by
photolyases (PL) with the aid of blue-light.
II. Phylogenetics
At this time, the photolyase/cryptochrome family has three members:
photolyase (cyclobutane pyrimidine dimer photolyase), (6–4) photolyase,
and cryptochrome. A number of phylogenetic trees based on sequence
comparisons of more than 100 members of the family across the three
BLUE-LIGHT PHOTORECEPTORS 75
Fig. 2. Structures of photolyase/cryptochrome cofactors. All photolyases

and cryptochromes contain FAD, and all either contain or are thought to contain
5,10-methenyltetrahydrofolate (MTHF) or 8-hydroxy-7,8-didemethyl-5-deazariboflavin
(8-HDF) as the second chromophore.
biological kingdoms have been generated (Brudler et al., 2003; Cashmore

et al., 1999), and a simple phylogenetic tree is shown in Figure 3. Some
interesting points that emerge from these analyses will be briefly summar-
ized. Photolyase has been found in many species from the prokaryotic,
eukaryotic, and archaeal kingdoms, and even in some viruses (Sancar,
2003; Srinivasan et al., 2001; Willer et al., 1999) (Table I). However, it has
also been found that many species from all three kingdoms lack the
enzyme. Prokaryotic organisms that possess the enzyme include Escherichia
coli and Bacillus firmus. Other prokaryotes, such as Haemophilus influenzae
and Bacillus subtilis, do not have photolyase. Among eukaryotic organisms
that are used as model systems, Saccharomyces cerevisiae contains photo-
lyase, but Schizosaccharomyces pombe does not. Of multicellular organisms,
Caenorhabditis elegans and garter snake lack photolyase, but rattlesnake,
zebrafish, and goldfish contain the enzyme. Many vertebrates appear to
have photolyase. Curiously, however, when mammals were separated into
marsupials and placentals, the former retained photolyase, and the latter
did not. Thus, opossum has photolyase but raccoons and humans do not.
76 SANCAR
Fig. 3. Phylogentic relationship among select members of the photolyase/

cryptochrome family. Sequences were aligned with Clustal W, and the tree was
produced by the Neighbor-Joining method using MEGA 2.1. Bootstrap confidence
values are shown (values for interior branches 95% are statistically significant). At,
Arabidopsis thaliana; Dm, Drosophila melangaster; Ec, Escherichia coli; Hs, Homo sapiens; Vc,
Vibrio cholerae.
Table I
Distribution of Photolyase and Cryptochromes in the Biological World
Enzyme/Photoreceptor
Photolyase (6–4) Photolyase Cryptochrome
Eubacteria
B. subtilis No No No
E. coli Yes No No
V. cholerae Yes No Yes(2)
Archaea
M. janaschii No No No
M. thermoautotrophicum Yes No No
Eukarya
S. pombe No No No
C. elegans No No No
S. cerevisiae Yes No No
D. melanogaster Yes Yes Yes
H. sapiens No No Yes (2)
A. thaliana Yes Yes Yes (3)
Viruses
Shoppe (rabbit) fibroma virus Yes No No
Fowlpox virus Yes No No
All plants tested so far, including Arabidopsis thaliana, tobacco, and soy-
bean, do have photolyase.
The (6–4) photolyase was first discovered in D. melanogaster (Todo et al.,
1993, 1996). Subsequently it was found in Xenopus laevis, rattlesnake (Kim
et al., 1994), zebrafish, and A. thaliana, among many other species (Todo,
1999). Of special interest, the enzyme has not been found in birds and
mammals (Hsu et al., 1996). Thus, humans lack both photolyase and (6–4)
photolyase and cannot carry out photorepair of UV-induced DNA damage.
They rely solely on nucleotide excision repair for eliminating these
potentially mutagenic and carcinogenic lesions from their DNA.
Cryptochrome was first discovered in plants (Ahmad and Cashmore,
1993; Malhotra et al., 1995), and subsequently in humans (Hsu et al.,
1996). It has since been found in organisms ranging from Vibrio cholerae
(Worthington et al., 2003) to Drosophila (Sancar, 2000; Todo, 1999) to
zebrafish, which contains six cryptochrome genes (Brudler et al., 2003;
Todo, 1999). It has been found in all insect and bird species tested. Of
organisms that are used as model systems, Drosophila has one cryptochrome,
humans and mice have two, and Arabidopsis has three cryptochromes.
Most bacteria including E. coli lack cryptochromes, Synechocystis possess
one (Brudler et al., 2003; Ng and Pakrasi, 2001), and V. cholerae has two
cryptochromes. Caenorhabditis elegans, which lacks photolyase and (6–4)
photolyase, also does not have a cryptochrome.
III. Structure of Photolyase

Photolyases are monomeric proteins of 450–550 amino acids and two
noncovalently bound cofactors (Johnson et al., 1988; Jorns et al., 1984).
One of the cofactors is always FAD. The other cofactor, which is also called
the second chromophore, is methenyltetrahydrofolate (MTHF) in the
majority of photolyases and 8-hydroxy-7,8-didemethyl-5-deazariboflavin
(8-HDF) in a limited number of species (some archaea, Anacystis nidulans,
and some ferns that synthesize this cofactor) (Eker et al., 1988, 1990;
Johnson et al., 1988; Kiener et al., 1989; Malhotra et al., 1992).
Crystal structures of photolyases from E. coli (Park et al., 1995),
A. nidulans (Tamada et al., 1997), and Thermus thermophilus (Komori et al.,
2001) have been solved. Although they possess different second chromo-
phores (E. coli photolyase has MTHF and the latter two contain 8-HDF),
and the level of overall sequence homology among the three enzymes is
only about 25% sequence identity, the structures of all three are remark-
ably similar. Here, the structure of E. coli photolyase will be presented and
the minor differences of the other two photolyases from this structure will
be briefly mentioned.
78 SANCAR
E. coli photolyase is made up of two well-defined domains (Fig. 4): an N-

terminal / domain (residues 1–132) and a C-terminal -helical domain
(residues 204–471). The two domains are connected to one another with a
long loop (residues 132–203) that wraps around the / domain (Fig. 4).
The MTHF photoantenna is located in a shallow cleft between the two
domains and is partially exposed to solvent. In contrast, the FAD cofactor
is deeply buried within the -helical domain and has the unusual U-
shaped (or cis) conformation, in which the flavin and the adenine rings
are stacked on top of one another. The flavin is accessible to the flat
surface of the -helical domain through a hole in the middle of this
domain. The hole has the right dimensions and polarity to allow the entry
of a thymine dimer to within van der Waals contact distance to the
isoalloxazine ring of FAD. Surface potential representation of the enzyme
reveals a positively charged groove running the length of the molecule and
passing through the entrance of the hole. These structural features led to
the suggestion that photolyase binds to the backbone of the damaged
strand and ‘‘flips’’ the cyclobutane dimer into the active site within the
hole so that high-efficiency electron transfer from the flavin to the pyrimi-
dine dimer can be effected by light (Park et al., 1995). The crystal structure
shows that the center-to-center distance between MTHF and FAD is 16.8 Å.
Surprisingly, the planes of the two chromophores, and hence presumably
Fig. 4. Structure of Escherichia coli photolyase. (A) Ribbon diagram representation.

The MTHF antenna is exposed on the surface, whereas the FADH catalytic cofactor is
buried within the core of the -helical domain. (B) Surface potential representation.
Blue, basic residues; red, acidic residues; white, hydrophobic residues. Note the
positively charged groove running diagonally the length of the protein and the hole
(marked by a square) with asymmetric charge distribution along the side walls and
leading to the flavin located in the bottom. (See Color Insert.)
the angle between their transition dipole moments, are nearly per-
pendicular to one another, which is not conducive for high-efficiency
energy transfer.
The structure of A. nidulans photolyase is very similar to that of the E. coli
enzyme, with one important exception: the 8-HDF photoantenna is deeply
buried into the interdomain cleft and the center-to-center distance
between the two chromophores is 17.5 Å. However, the planes of the two
chromophores are nearly parallel, allowing for more efficient energy
transfer from the second chromophore to FAD, even though they are farther
apart than the two chromophores in E. coli photolyase (Kim et al., 1992;
Tamada et al., 1997). The T. thermophilus photolyase has a shorter interdo-
main loop and is overall more compact, with more extensive interdomain
contacts between the two domains, consistent with its thermostability
(Komori et al., 2001).
IV. Reaction Mechanism of Photolyase

The reaction mechanism of photolyase has been investigated in
considerable detail (Sancar, 1994, 2003). In classical enzymological ter-
minology, photolyase performs catalysis by a ‘‘sequential ordered mech-
anism’’ (Fig. 5): the enzyme must bind to one substrate (Pyr<>Pyr) first
before it can bind (absorb) the second substrate (a photon) and carry
out catalysis. In contrast to all other enzymes, however, the second sub-
strate is not another molecule, but a photon that can excite the cofactors
of the enzyme (binding equivalent) in a femtosecond. This unique prop-
erty of photolyase has been used advantageously to analyze the various
kinetic steps both in vivo and in vitro by carrying out the binding under
yellow light that does not excite the enzyme, and then delivering the
photoreactivating photon in light pulses of duration ranging from
20 fs to 1 ms (Kim et al., 1991; Langenbacher et al., 1997; MacFarlane
and Stanley, 2003). Remarkably, when the kinetic constants for the
reaction
k1
EþSÐ h
ES !
kp
EþP
k 2
obtained by flash photolysis in vivo and in vitro are compared, the agree-
ment between the two sets of values is excellent (Sancar et al., 1987). Such
a comparison can be made for only a very limited enzyme system at
present, and the results obtained by photolyase validate the relevance of
in vitro thermodynamic and kinetic parameters to the in vivo reactions.
In the following text, we will analyze the binding of and catalysis by
80 SANCAR
Fig. 5. Reaction mechanism of photolyase. (A) Binding (dark reaction). The

enzyme binds DNA containing a T<>T by random collision and flips out the dimer
into the active site pocket. Enzyme–substrate complex formation is a thermal reaction
(kT), independent of light. Following the light (hv) reaction, the repaired dinucleotide
is ejected from the active site cavity and DNA dissociates from the enzyme. (B) Catalysis
(light reaction). The photoantenna chromophore MTHF absorbs a photon and
transfers the excitation energy to FADH, which then transfers an electron to T<>T to

generate a biradical. The cyclobutane ring is split, and the electron returns to FADH to
regenerate catalytically active FADH. The repaired thymine dinucleotide is extruded
from the active site, and the enzyme dissociates from DNA. (See Color Insert.)
photolyase separately because these two events are temporally separated

under natural reaction conditions.
A. Binding of Photolyase to Substrate

Photolyase binds to Pyr<>Pyr in DNA with a second-order rate constant
(kon 108107 M1 sec1) consistent with target location by three-dimen-
sional diffusion; there is no evidence for a diffusion-controlled reaction in
reduced dimensionality (Husain and Sancar, 1987), as has been found for
some sequence-specific DNA binding proteins. The specific binding con-
stant to a T<>T in DNA is KS ¼ 109 M, and the nonspecific binding
constant for a dinucleotide in undamaged DNA is KNS 104 M (Husain
and Sancar, 1987). Thus, the selectivity factor of the enzyme for Pyr<>Pyr
is KS/KNS 105. This high selectivity is achieved by the somewhat unique
backbone structure of DNA containing a Pyr<>Pyr, and by the dinucleo-
tide flipping of Pyr<>Pyr in the active site in preference to a Pyr–Pyr
dinucleotide. Finally, absolute specificity is conferred by the chemical
proofreading step, whereby the excited-state flavin can donate an electron
to a Pyr<>Pyr but not to a nondamaged base that may happen to be in the
active site.
The presence of a T<>T in a duplex causes 9 degree unwinding and
about 30 degree kinking into the major groove, as revealed by both
solution (Husain et al., 1988) and x-ray crystallographic analyses (Park
et al., 2002). The crystal structure of a decamer duplex with a T<>T was
solved recently and is shown in Figure 6. This unique structure of the
duplex, and in particular that of the damaged strand with which photo-
lyase makes nearly all of its contacts (Husain et al., 1987), provides a
considerable degree of specificity. However, similar but not identical
backbone distortions are caused by other DNA lesions, and as a conse-
quence, such lesions that are not repairable by photolyase can constitute
high-affinity binding sites. For example, E. coli photolyase binds to cisplat-
in-d(GpG) diadduct with affinity close to its affinity for a Pyr<>Pyr, and it
stimulates the repair of both lesions by nucleotide excision repair (Sancar
et al., 1984; Özer et al., 1995). Thus, backbone distortion is an important
but insufficient structural determinant of specific binding of photolyase to
a Pyr<>Pyr in DNA. The second level of specificity is achieved by what
might be called an induced-fit mechanism. It has been proposed that
photolyase, in a manner similar to DNA methyltransferases (Roberts and
Cheng, 1998), ‘‘flips out’’ its substrate from within the duplex to the active
site in the enzyme (Park et al., 1995). The size and distribution of the
polar, charged, and aromatic residues within the hole leading to flavin are
such that only a cyclobutane pyrimidine dimer can be accommodated
82 SANCAR
Fig. 6. Schematic diagram illustrating the kink induced in DNA by T<>T (Park
et al., 2002). Regular B-DNA decamer and a T<>T containing decamer are depicted in
green and blue, respectively. The thymines making up the cyclobutane dimer are drawn
in red. The view (A) shows part of the major groove, and the view (B) shows the minor
groove of the duplex. The phosphodeoxyribose backbone shows a sharply kinked or
pinched structure (courtesy of Dr. ChulHee Kang). (See Color Insert.)
within the active site. As a consequence, the enzyme can flip out only a
pyrimidine dimer into the active site hole, and even though a cocrystal
structure is not available at present, most likely some conformational
change in the enzyme itself and in the dimer occurs during dinucleotide
flipping to optimize the flavin–dimer contacts. It must be noted that
because of the loss of aromaticity, the pyrimidine moieties of the dimer
are no longer planar and have lost stacking interactions. As a conse-
quence, a Pyr<>Pyr is structurally quite different from a Pyr–Pyr dinucle-
otide, and the latter most likely cannot be flipped out into the active site
cavity. Moreover, the Pyr–Pyr dinucleotide that forms following repair no
longer fits into the cavity and is ejected from the active site.
The binding of photolyase to its substrate has been extensively investi-
gated with DNase and chemical footprinting methods (Baer and Sancar,
1989; Husain et al., 1987; Kiener et al., 1989) and with substrates ranging in
size from 2 to 50 nt (Husain et al., 1987; Jorns et al., 1984; Kim and Sancar,
1991). These investigations have revealed that photolyase binds to a T<>T
within ssDNA and dsDNA with equal affinity and that it contacts the
phosphate 50 to the T<>T and the three phosphates 30 to the dimer,
but not the intradimer phosphate. The enzyme has essentially the same
affinity for a substrate in the form of NpT<>TpNpNpN as for a substrate
of about 50 bp with a T<>T. Thus, the hexameric substrate has all
the structural determinants necessary for high-affinity and high-specificity
binding. However, photolyase binds to a T<>T as well as other Pyr<>Pyr
dinucleotides with a KD 105M, indicating that the cyclobutane pyrimi-
dine dimer itself contributes about half of the binding free energy. Thus, it
appears that half of the binding free energy is contributed by the interac-
tion of the positively charged groove on the enzyme with the distorted
backbone of the damaged strand, and the other half is provided by
dinucleotide flipping into the active site cavity where ionic, stacking,
and van der Waals interactions contribute to the stability and specificity
of the complex.
To recapitulate, photolyase locates Pyr<>Pyr by three-dimensional dif-
fusion, and the positively charged groove on the enzyme surface makes a
low-specificity complex by ionic interactions with the 30-degree kinked
damaged strand causing further distortion, resulting in the flipping out of
the dimer into the active site cavity, lined at the bottom with flavin and at
the sides with two tryptophans. This drastic conformational change leads
to the development of a new set of interactions between the enzyme and
substrate and the formation of a stable and specific complex. However, the
ultimate specificity is achieved at the chemical step: Even if an undamaged
dipyrimidine or another DNA lesion were to be placed in the active site, as
far as is known, the enzyme can transfer an electron only to cyclobutane
pyrimidine dimers, and hence the chemical step provides near-absolute
specificity of photolyase for Pyr<>Pyr.
B. Catalysis by Photolyase
Photolyase catalyzes light-initiated (
s2 þ
s2) cycloreversion of the
cyclobutane ring joining the two pyrimidine moiety in a pyrimidine dimer.
Catalysis occurs by a photo-induced cyclic electron transfer reaction that
does not cause a net change in the redox state of the enzyme and
substrate/product at the end of the catalytic cycle (Li et al., 1991; Payne
and Sancar, 1990; Sancar, 2003). The basic features of catalysis are as
follows (Fig. 5B): A 300–500-nm photon is absorbed by MTHF (or 8-HDF).
The excited MTHF singlet, 1MTHF*, transfers energy by fluorescence
resonance energy transfer to FADH to generate 1(FADH)*, which
84 SANCAR

within 50 ps transfers an electron to Pyr<>Pyr to generate the FADH -

Pyr<>Pyr biradical. The dimer radical splits to two canonical pyrimi-
dines concomitant with back electron transfer within 0.5–2 ns to FADH to
restore it to the catalytically competent FADH form. The splitting of the
cyclobutane ring is thought to be by a concerted but asynchronous cleav-
age of the C5–C5 and C6–C6 bonds of the cyclobutane ring. Splitting of
the dimer causes a considerable change in the structure of the dinucleo-
tide that makes up the dimer and in the structure of the DNA backbone
in the immediate vicinity of the dimer. As a consequence, the two
pyrimidines are ejected from the active site cavity, the interaction of the
positively charged groove on the photolyase surface with the DNA back-
bone weakens, and the enzyme dissociates from DNA to enter new rounds
of catalysis. The photochemical reactions from absorbing a photon by

MTHF to splitting of Pyr<>Pyr and restoration of FADH to FADH by
back-electron transfer are very fast and are expected to be completed
within 0.5–2.0 ns to close the photocycle. The substrate binding and
product dissociation reactions are relatively slower than the photochemi-
cal reaction and therefore are the rate-determining steps in the overall
catalytic cycle.
1. Quantum Yield
Quantum yield, in photochemical reactions, is the ratio of the number of
chemical reactions caused by light to the number of photons absorbed by
the chemical species. With the exception of some rare photochemical
processes in bio-inorganic chemistry, in which chain reactions initiated by
absorption of a single photon result in multiple catalytic events and hence
quantum yield greater than unity, in the vast majority of photochemical
reactions and in all known photobiological reactions such as photosynthesis,
vision, and phototropism the quantum yield is less than 1.0.
The quantum yield of DNA repair by photolyase (the number of cyclo-
butane pyrimidine dimers split by the enzyme for each photon absorbed
by the enzyme in the enzyme-substrate complex) ranges from 0.7 to 1.0.
It should be noted, however, that in photolyase FADH is the catalytic
cofactor and MTHF (or 8-HDF) is the photoantenna. As a consequence,
the quantum yield of photolyase is the product of three reactions (Payne
and Sancar, 1990): energy transfer from 1MTHF* (or 8-HDF) to FADH,
electron transfer from 1(FADH)* to the Pyr<>Pyr, and finally splitting of

Pyr<>Pyr . The latter two reactions are very efficient and occur with
nearly 100% efficiency, at least in the case of T<>T. Therefore, the critical
determinant of overall quantum yield of repair is the quantum yield of
energy transfer from the photoantenna to the catalytic cofactor (Kim et al.,
1991, 1992). The efficiency of energy transfer by Förster radiationless
transfer mechanism is inversely proportional to the distance between the

donor and the acceptor, and to the angle between the transition dipole
moments of the donor and acceptor. Optimum efficiency is achieved
when the interchromophore distance is short and the transition dipole
moments are parallel. The interchromophore distance in E. coli photo-
lyase is 16.8 Å, and in the A. nidulans photolyase it is 17.5 Å. However,
despite the greater distance between the chromophores in A. nidulans,
photolyase energy transfer from 8-HDF to FADH occurs with nearly 100%
efficiency because the planes of the two chromophores (and hence pre-
sumably the transition dipole moments) are nearly parallel. In contrast,
in E. coli photolyase, the transition dipole moments of MTHF and FADH
are nearly perpendicular to one another, and as a consequence, energy
transfer from MTHF to FADH occurs with 70%–75% efficiency. Be-
cause the quantum yields for subsequent reactions for both enzymes are
identical and near unity (Kim et al., 1991, 1992), the efficiency of inter-
chromophore energy transfer determines the overall quantum yield of
repair. Thus, for E. coli photolyase and other folate class photolyases, the
overall quantum yield of repair is 0.7–0.75 (Malhotra et al., 1994; Payne
and Sancar, 1990), and that for deazaflavin class enzymes is very close to
1.0; that is, for every photon absorbed by the enzyme, one pyrimidine
dimer is repaired.
2. Action Spectrum
An action spectrum is a plot of the rate of a photochemical reaction as a
function of the wavelength of light effecting the reaction. In general, the
action spectrum has the shape of the absorption spectrum of the photo-
active pigment catalyzing the reaction. In photolyase, an enzyme with
FADH and no MTHF (or 8-HDF) is capable of repairing DNA, albeit
less efficiently than the holoenzyme (Kim et al., 1992; Payne and Sancar,
1990), but enzymes containing MTHF (or 8-HDF) but no FADH are
catalytically inert (Kim et al., 1991, 1992). Despite this central role of
FADH in catalysis, under physiological conditions, more than 90% of
the photons used for catalysis are absorbed by MTHF (or 8-HDF), and the
absorption spectrum of the second chromophore determines the shape of
the action spectrum of photolyase for two reasons. First, the FADH has an
absorption maximum around 360 nm and an extinction coefficient of
5,000 M1 cm1 at this wavelength. In contrast, MTHF and 8-HDF have
much higher extinction coefficients and absorb at longer wavelengths:
The extinction coefficient of MTHF is 25,000 M1 cm1, and its absorp-
tion maximum ranges from 377 to 415 nm, depending on the particular
enzyme; the extinction coefficient of 8-HDF is 44,000 M1 cm1, and its
absorption maximum is at 440 nm. Second, the fraction of photons in
86 SANCAR
Fig. 7. Absorption and action spectra of DNA photolyases. Left, Escherichia coli
photolyase. Solid and broken lines represent the absorption spectra of the E-MTHF-
FADH and the E-FADH forms of the enzyme, and the triangles and squares represent
the photolytic cross sections (x) of the two forms. Right, Anacystis nidulans photolyase.
The solid and broken lines are the absorption spectra of the E-8-HDF-FADH and the
E-FADH forms of the enzyme, and the circles and triangles represent photolytic cross
sections of the corresponding forms at selected wavelengths.
sunlight in the 300–350-nm range reaching the earth surface is very low
compared to those >350 nm. As a consequence, most repair in nature is
mediated by photons absorbed by the second chromophore, even though a
photon absorbed directly by FADH is certainly more efficient in photo-
repair. As a general rule, MTHF class photolyases have an essentially
symmetrical action spectra, with max 375–415 nm, and those in the
8-HDF class have an action spectrum nearly identical to the absorption
spectrum of enzyme-bound 8-HDF, with a peak at 444 nm (Fig. 7).
V. (6–4) Photolyase
The (6–4) photoproduct is the second most abundant lesion induced in
DNA by UV light, constituting 10%–20% of total UV photoproducts
(Taylor, 1994). In contrast to cyclobutane pyrimidine dimers that are
formed from the excited triplet state of pyrimidines, the (6–4)
photoproducts are formed from the pyrimidine excited singlet state. In
the (6–4) photoproduct, the C6 of the 50 pyrimidine makes a sigma bond
with the C4 of the 30 pyrimidine, and the OH (or NH2) group at the C4
of the 30 pyrimidine is transferred to the C5 of the 50 pyrimidine. As

a consequence, breaking the C6–C4 sigma bond either thermally or
photochemically does not repair the DNA lesion but actually converts
a dinucleotide adduct to two adjacent damaged bases (see Fig. 1B). The
(6–4) photoproduct distorts the DNA more severely than the cyclobutane
dimer and is recognized and repaired by both the bacterial (Svoboda et al.,
1993) and the human (Reardon and Sancar, 2003) excision nuclease
systems five- to 10-fold more efficiently than the cyclobutane dimer
pyrimidine dimer. The classical photolyase neither recognizes nor repairs
the (6–4) photoproduct (Brash et al., 1985). However, there is a (6–4)
photoproduct-specific photolyase that reverses this lesion in a light-
dependent reaction (Todo et al., 1993, 1996). The (6–4) photolyase was
first discovered in Drosophila (Todo et al., 1993) and was subsequently
found in many other species (Chen et al., 1994; Kim et al., 1994; Todo,
1999). The (6–4) photolyases exhibit a high level of sequence identity to
photolyase, and those that have been characterized biochemically appear
to contain both chromophores (Zhao et al., 1997). However, as the enzyme
has been isolated only as recombinant protein expressed in heterologous
sources, often the cofactors are present at substoichiometric levels. Thus,
the X. laevis (6–4) photolyase expressed in E. coli contains nearly
stoichiometric FAD but no detectable folate (Hitomi et al., 1997). Similar-
ly, the D. melanogaster (6–4) photolyase expressed in E. coli contains both
FAD and folate, but the former occurs at a stoichiometry of 0.01–0.05, and
the latter at even lower levels relative to the apoenzyme (Zhao et al., 1997).
To date, (6–4) photolyase has not been found in organisms that synthesize
5-deazaflavin, and hence there is no evidence for the presence of (6–4)
photolyases that use 8-HDF or any chromophore other than folate as a
photoantenna.
A. Binding of (6–4) Photolyase

The Drosophila and Xenopus (6–4) photolyases appear to bind DNA con-
taining a (6–4) photoproduct by three-dimensional diffusion and to make
contacts around the lesion quite similar to the contacts made by
photolyase with DNA containing a cyclobutane pyrimidine dimer (Hitomi
et al., 1997; Zhao et al., 1997). The (6–4) photolyase, like the cyclobutane
photolyase, binds to its cognate lesion in ssDNA and dsDNA with
essentially equal affinities (Zhao et al., 1997). When bound to a dsDNA
substrate, the enzyme confers single-strandedness to a 4-bp region around
the lesion, and the presence of a mismatch across the (6–4) photoproduct
increases the affinity of the enzyme for the substrate (Zhao et al., 1997).
These three features of binding, that is, binding to substrate in ssDNA with
88 SANCAR
high affinity, conferring single-strandedness to the bases immediately

around the target, and binding with higher affinity when the target base
is in the context of a mismatch, are standard criteria for a base-flipping
mechanism (Roberts and Cheng, 1998), and hence it was proposed that
(6–4) photolyase, like classical photolyase, employs a dinucleotide flipping
mechanism to achieve specificity following the low-specificity interactions
with the distorted DNA backbone at the site of the lesion (Zhao et al.,
1997). Indeed, a molecular modeling study of Xenopus (6–4) photolyase
using the E. coli photolyase C backbone as a template revealed a positively
charged groove on the surface of the enzyme and a pocket in the center of
the groove leading to the FADH in the core of the -helical domain, and
the cavity appears to have the appropriate size and charge distribution to
accommodate a (6–4) photoproduct (Todo, 1999). The equilibrium-
binding constants of (6–4) photolyases of Drosophila and Xenopus are in
the range of KD ¼ 0.5–1.0 109 M, and the dissociation rate constant is
koff 103 s1 to 105 s1 (t1/2 10 to 100 min). Thus, it has been
concluded that the formation of the enzyme–substrate complex is diffusion
controlled and the main determinant of high specificity is the slow off rate
of dissociation of the enzymes from complexes formed at the damage
site. The dissociation rate following repair has not been determined, but
it is expected to be much faster than that of unrepaired substrate. Increasing
the off rate several orders of magnitude would still be much slower than the
photochemical reaction, which is most likely complete within a nanosecond
or less. Hence, under substrate saturating (damage and photon) conditions
for (6–4) photolyase, as in the case of photolyase, the rate-determining step
in the overall reaction is the dissociation of the repaired product.
B. Catalysis by (6–4) Photolyase

Catalysis by (6–4) photolyase must accomplish two chemical tasks: cleav-
age of the C6–C4 sigma bond, and transfer of the OH (or NH2) group
from the C5 of the 50 base to the C4 of the 30 base. Because formation of
the (6–4) photoproduct is presumed to proceed through a four-mem-
bered oxetane or azetidine intermediate, it has been proposed that (6–4)
photolyase first converts the ‘‘open’’ form of the (6–4) photoproduct to
the four-membered ring by a thermal reaction, and then the four-mem-
bered ring is cleaved by retro [2+2] reaction photochemically (Kim et al.,
1994; Zhao et al., 1997). A site-directed mutagenesis study has identified
two histidine residues in the active site that may participate in conversion
of the (6–4) photoproduct to the oxetane intermediate by general acid–
base catalysis (Hitomi et al., 2001). A current model for catalysis by (6–4)
photolyase is as follows (Fig. 8): The enzyme binds DNA and flips out the
Fig. 8. Reaction mechanism of (6–4) photolyase. The enzyme binds to DNA

containing a (6–4) photoproduct and flips out the dinucleotide adduct into the active
site cavity, where the ‘‘open’’ form of the photoproduct is converted to the oxetane
intermediate by a light-independent general acid-base mechanism. Catalysis is initiated
by light; MTHF absorbs a photon and transfers energy to FADH, which then transfers
an electron to the oxetane intermediate; bond rearrangement in the oxetane radical
regenerates two canonical pyrimidines, and back-electron transfer restores the flavin
radical to catalytically competent FADH form. The repaired dipyrimidine flips back
into the DNA duplex, and the enzyme is dissociated from the substrate.
(6–4) photoproduct into the active site cavity, where the photoproduct is
converted into the oxetane form thermally. A 350–450-nm photon is
absorbed by the folate photoantenna, which transfers energy to FADH.
The 1(FADH)* transfers an electron to the oxetane ring initiating the
cycloreversion reaction, which is followed by back-electron transfer
to restore the flavin radical (Zhao et al., 1997). This is a plausible model;
however, at present, direct evidence for energy transfer from the
photoantenna to flavin is lacking. Evidence for electron transfer from
flavin to substrate was obtained by demonstration of a requirement for
reduction of flavin either chemically or photochemically for catalysis
(Hitomi et al., 1997; Zhao et al., 1997). Strong support for the proposed
mechanism was provided by a study with a model system (Cichon et al.,
2002): an oxetane ring was covalently linked to flavin, and its cleavage by
90 SANCAR
light was investigated under a variety of conditions. It was found that only
two-electron reduced and deprotonated flavin induced photosplitting of
the oxetane ring at a significant rate.
Clearly, all indications are that (6–4) photolyase binds DNA and repairs
its substrate by a mechanism quite similar to that of classical photolyase.
However, there appears to be a fundamental difference in the photochem-
ical reaction catalyzed by the two enzymes. The quantum yield of repair by
excited singlet-state flavin by classical photolyase is near unity, whereas the
quantum yield of repair by excited flavin in (6–4) photolyase is 0.05–0.10.
Whether this low quantum yield of repair by (6–4) photolyase is a result
of the low efficiency of formation of the oxetane intermediate thermally,
low efficiency of electron transfer from the flavin to the photoproduct,
or low efficiency splitting of the oxetane anion coupled with high rate of
back electron transfer is not known at present. Furthermore, it was found
that (6–4) photolyase can photorepair the Dewar valence isomer of the
(6–4) photoproduct (Taylor, 1994) that cannot form an oxetane interme-
diate, casting some doubt about the basic premise of the retro [2þ2]
reaction. However, the Dewar isomer is repaired with 300–400 lower quan-
tum yield than the (6–4) photoproduct, and it has been proposed (Zhao
et al., 1997) that the Dewar isomer may be repaired by the enzyme through a
two-photon reaction in which the first photon converts the Dewar isomer to
the Kekule form and a second electron transfer reaction initiated by the
second photon promotes the retro [2þ2] reaction.
VI. Cryptochrome
Cryptochrome was originally used as a generic term for blue-light
photoreceptors that were known to exist and to regulate a variety of light
responses in plants but whose identities remained cryptic for over a
century. At present, at least three blue-light-specific receptors have been
identified in plants including phototropin, FKF1, and a receptor related to
photolyase (Briggs and Huala, 1999; Cashmore, 2003; Sancar, 2000). The
photolyase-like receptor was the first blue-light receptor identified in
plants and hence was called cryptochrome (Lin et al., 1996). When two
photolyase-like proteins with no photolyase activity were discovered in
humans, it was suggested that these may function as blue-light
photoreceptors that regulate the circadian clock, and they were named
cryptochrome 1 and cryptochrome 2 as well (Hsu et al., 1996). At pre-
sent, the term ‘‘cryptochrome’’ has acquired a precise meaning: a photo-
lyase-like protein with no DNA repair activity but with known or presumed
blue-light receptor functions (Sancar, 2000).
Cryptochrome has been found in bacteria, plants, and animals

(Cashmore, 2003). Its role in bacteria is not known (Brudler et al., 2003; Ng
and Pakrasi, 2001; Worthington et al., 2003), though it regulates growth and
development in plants (Cashmore, 2003; Lin and Shalitin, 2003) and the
circadian clock in animals (Sancar, 2003; Thompson and Sancar, 2002). Both
the circadian clock and DNA repair play a role in ameliorating the harmful
effects of sunlight. Thus, it is conceivable that in the distant past, when more
UV light reached the surface of the earth, an ancestor of modern lifeforms
contained a photoactive flavo-protein that acted as a photosensory pigment
that regulated the movement of the organisms from the water surface to a
depth inaccessible to UV, and at the same time repaired the UV-induced DNA
lesions that may have been induced before escape. Apparently, during evolu-
tion, these two functions diverged so that in present-day organisms, photore-
activation and circadian photoentrainment are mediated by evolutionarily
and structurally related blue-light photoreceptors called photolyase and
cryptochrome, respectively (Sancar et al., 2000).
A. Structure
Cryptochromes exhibit 20%–40% sequence identities to photolyases
(Cashmore et al., 1999; Todo, 1999). With the exception of V. cholerae
cryptochrome 1 (Worthington et al., 2003), all cryptochromes character-
ized to date have only been isolated by expressing the cryptochrome genes
in heterologous systems, mainly in E. coli (Hsu et al., 1996; Malhotra et al.,
1995) and in insect cells (Lin et al., 1996). When heterologously expressed
cryptochromes are purified from such sources, they contain very little or
no folate and usually substoichiometric flavin in an oxidized state. The
only exception is V. cholerae cryptochrome 1, which, when purified as a
recombinant protein expressed in either E. coli or in V. cholerae, contains
both the folate and the flavin chromophores in essentially one-to-one
stoichiometry with the apoenzyme and the flavin in the two-electron
reduced state (Worthington et al., 2003). The biochemical properties
of VcCry1 are the strongest evidence to date that cryptochromes may
function in a manner analogous to photolyases.
The crystal structure of the Synechocystis cryptochrome obtained by
molecular replacement, using the E. coli photolyase as a template (Brudler
et al., 2003), and the three-dimensional structure of human cryptochrome
2 obtained by molecular modeling onto the C backbone of E. coli
photolyase (Özgür and Sancar, 2003) reveals a basically photolyase-like
structure including, somewhat surprisingly, the positively charged groove
involved in binding to DNA and the hole in the middle of this groove
leading to the flavin in the core of the molecule (Fig. 9). It appears,
92 SANCAR
Fig. 9. Model for human cryptochrome 2. The model was computer generated
using the Escherichia coli DNA photolyase as a template; the C-terminal 80 amino acids of
hCRY2 were excluded. Left, ribbon representation. Right, surface potential representa-
tion. Note the presence of the positively charged groove on the surface and passing
through the hole leading to the FAD cofactor in the core of the -helical domain. (See
Color Insert.)
however, that the FAD in cryptochromes is more accessible to solvents

than the FAD of photolyases.
Most cryptochromes contain 20–200–amino acid C-terminal extensions
beyond the photolyase homology region. Interestingly, it has been found
that expression of this C-terminal domain of Arabidopsis cryptochromes
confers a constitutive ‘‘light-on’’ phenotype (Yang et al., 2001). Thus, it
appears that the C terminus acts as the effector function that is somewhat
repressed by the photolyase-like region in the dark and relieved in the light.
B. Function of Cryptochrome
In contrast to photolyases, the photochemical reaction carried out
by cryptochrome is not known. As a consequence, despite overwhelming
genetic evidence that cryptochromes function as photoreceptors, a legiti-
mate argument can be made that cryptochromes are simply molecules
involved in phototransduction but not photoreception (Sancar, 2000;
Van Gelder, 2002). The following reactions have been detected by in vivo
and in vitro biochemical experiments: first, A. thaliana CRY 2 but not CRY 1 is
degraded on exposure to light (Lin and Shalitin, 2003), and presumably the
same is also true for D. melanogaster cryptochrome (Stanewsky et al., 1998).

Second, human and Drosophila cryptochromes bind to their cognate clock
proteins called Tim and Per (see Reppert and Weaver, 2002). It was reported
that dCry-dTim interaction in the yeast two-hybrid system was light depen-
dent (Griffin et al., 1999) but that the interactions of the human crypto-
chromes with Per1 and Per2 proteins in the same system were light
independent. It was therefore suggested that the Drosophila, but not the
human cryptochrome, has a photoreceptor function (Ceriani et al., 1999).
However, when considering the fact that human cryptochromes expressed
in heterologous systems contain either grossly substoichiometric or no
cofactors, these findings are open to alternative interpretations. Indeed, a
more recent study found that dCry lacking the C-terminal 20 amino acids
interacts with both dTim and dPer in a light-independent manner in the
yeast two-hybrid assay (Rosato et al., 2001). Thus, it appears that although the
yeast two-hybrid system is useful in detecting protein-protein interactions, it
is prone to artifacts when used for proteins with intrinsic chromophores.
Third, it has been reported that AtCRY 1, At CRY 2, and human CRY 1 are
serine/threonine-specific protein kinases, and moreover, the kinase activity
of Arabidopsis cryptochromes was strongly stimulated by light (Bouly et al.,
2003; Shalitin et al., 2003). These are intriguing findings; however, how
autophosphorylation may initiate or regulate signal transduction is unclear.
Indeed, many years after the discovery of autophosphorylating kinase
activities of two classes of light receptors in plants, phytochrome and
phototropin, the significance of the kinase activities of these proteins to
their photoreception/phototransduction functions remains controversial
(Briggs and Huala, 1999). Fourth, it has been found that human CRY 2
interacts with serine/threonine phosphatase PP5 and modulates its activity
(Zhao and Sancar, 1997). Similarly, Arabidopsis CRY 1 interacts with the PP7
serine/threonine phosphatase PP7, and this interaction is necessary for
blue-light response (Moller et al., 2003). Fifth, cryptochromes bind to nu-
cleic acids. Human CRY 2 binds to DNA with modest affinity and with higher
affinity to DNA containing a (6–4) photoproduct; however, binding to the
photolesion, in contrast to photolyases, is not affected by light (Özgür and
Sancar, 2003). Similarly, V. cholerae cryptochrome 1 binds to RNA
(Worthington et al., 2003), although the significance and specificity of this
binding is unknown at present.
In contrast to the paucity of biochemical data on the photosensory func-
tions of cryptochromes, there are extensive genetic and cell biology data on
the roles of cryptochromes in blue light, photoreception in plants and
animals, and circadian clock regulation in animals (Cashmore, 2003; Lin
and Shalitin, 2003; Sancar, 2003). In Arabidopsis, blue light inhibits elonga-
tion of hypocotyls in a cryptochrome-dependent manner. In animals,
94 SANCAR
cryptochromes were first discovered in humans and were hypothesized to be

the photosensory pigment for regulating the circadian clock (Hsu et al.,
1996). Indeed, both mammalian cryptochromes are expressed at relatively
high levels in the inner retina, a region capable of synchronizing the
circadian clock with the daily light–dark cycle, independent of the outer
retina that is required for vision (Miyamoto and Sancar, 1998; Thompson
et al., 2003) (Fig. 10A). Both cryptochromes are also expressed in all mam-
malian cells at moderate levels, and mCRY 1 is expressed at a particularly
high level in the master circadian pacemaker in an area in the hypothalamus
above the optic nerve, called the suprachiasmatic nuclei (SCN). Moreover,
the expression of both CRY 1 and CRY 2 exhibits circadian (daily) rhythmic-
ity, peaking at about 2:00 p.m. in the SCN and reaching a nadir at about 2:00
a.m.; expression in peripheral organs is 4–6 hours out of phase with that
of the SCN expression (Miyamoto and Sancar, 1999). Mice lacking crypto-
chromes are seriously compromised in photoreception/phototransduction
to the SCN (Selby et al., 2000; Thompson et al., 2004; Thresher et al.,
1998; Vitaterna et al., 1999) but are not circadian blind because of func-
tional redundancy between the cryptochromes and opsins that are ex-
pressed in the outer and inner retina. Mutation in the sole Drosophila
cryptochrome has a similar effect on circadian photoreception in this
organism (Stanewsky et al., 1998), consistent with functional redundancy
of opsins and cryptochromes in circadian photoreception.
In addition to their light-dependent effect of unknown mechanism,
mammalian cryptochromes have a light-independent function that is nec-
essary for normal functioning of the circadian clock. Thus, wild-type mice
kept in constant darkness maintain a circadian rhythm of activity and rest
phases with 23.7 hour periodicity. In mice lacking Cry2, the period is longer,
at 24.7 hours (Thresher et al., 1998); in mice lacking Cry1 the period is
shorter, 22.5 hours (Van der Horst et al., 1999; Vitaterna et al., 1999); and
mice lacking both cryptochromes are arrhythmic (Van der Horst et al., 1999;
Vitaterna et al., 1999). Co-immunoprecipitation and reporter gene assays
have led to a considerable insight into the ‘‘clock function’’ of the crypto-
chrome. In mice, the molecular clock, which engenders the behavioral
clock, is made up of transcription factors CLOCK, BMAL1, PER1, PER2,
and CRY 1 and CRY 2 (Reppert and Weaver, 2002) (Fig. 10B). CLOCK and
BMAL1 make a heterodimer that binds to the E-box in the promoters of PER
and CRY genes and stimulate their transcription; the CRY and PER proteins
make combinatorial heterodimers in the cytoplasm, which translocate into
the nucleus and bind to the CLOCK protein, interfering with its activator
function. Because of necessity for posttranslational modification of the
various components of the molecular clock and the delay between protein
synthesis and nuclear translocation, the transcription-stimulating activity
Fig. 10. Circadian photoreception and the molecular clock in mammals. (A)
Circadian and visual photoreception/phototransduction in mammals. Light signal
received by the rods and cones in the outer retina is transmitted to the visual cortex by
the optic nerve (blue). Light signal received by cryptochromes and inner retinal opsins
is transmitted to the circadian center in the midbrain, an area called the
suprachiasmatic nuclei (SCN) by a subset of the optic nerve fibers (red). (B) The
molecular clock. The transcription factors clock, and BMal1 make a heterodimer that
acts on the promoter of Cry and Per genes activating their transcription. The CRY and
Per proteins heterodimerize in the cytoplasm, undergo posttranslational modification,
and translocate into the nucleus, where they interfere with the CLOCK-BMAL1 activity
and repress their own transcription as well as those of other genes regulated by clock-
BMal1. The transcriptional activation/inhibition cycle has a period of about 24 hours
resulting in daily oscillation of clock-controlled functions. (See Color Insert.)
96 SANCAR
of the BMAL1-CLOCK heterodimer exhibits periodicity of about 24

hours. In the absence of cryptochromes, there is no inhibition of
BMAL1-CLOCK heterodimer, which causes constitutively high levels of
clock gene transcripts (Vitaterna et al., 1999), resulting in molecular and
behavioral arrhythmicity. As is apparent from this summary, the light-
independent function of cryptochrome is well understood in animals,
but the photoreceptive function remains ill defined.
VII. Conclusion
The photolyase/cryptochrome blue-light photoreceptor family encom-
passes a large group of proteins from all three biological kingdoms. These
proteins absorb near-UV/blue light and use the light energy to repair far
UV-induced DNA damage or to reset the circadian clock. Both photolyase
and cryptochrome also perform light-independent functions in DNA
repair and in generating the molecular circadian clock, respectively. In
addition, cryptochromes regulate blue-light-dependent growth and
development in plants. Finally, cryptochromes have now been identified
in the nonphotosynthetic bacterium, V. cholerae, which has no known
photoresponses. Future work is likely to uncover novel light-dependent
and light-independent functions mediated by cryptochromes.
Acknowledgments
I thank my student Carrie L. Partch for her critical comments on the manuscript and for
preparing the figures. I am grateful to Professor ChulHee Kang and Dr. H. Park for providing
Figure 6. This work was supported by NIH grant GM31082.
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COORDINATION OF REPAIR, CHECKPOINT, AND CELL
DEATH RESPONSES TO DNA DAMAGE
By JEAN Y. J. WANG AND SARAH K. CHO
Division of Biological Sciences and the Moores Cancer Center,

University of California, San Diego,
La Jolla, California 92093
I. Introduction . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 101
II. Overview of Biological Responses to DNA Damage. .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 104
A. General Framework of DNA Damage Responses. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 104
B. Temporal Coordination of DNA Damage Responses. . . . . . . . . . . . . . . . . . . .. . . . . . 104
C. Other Comments on the General Framework . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 109
III. Molecular Components for the Initiation of DNA Damage Responses. . . .. . . . . . 109
A. DNA Damage Sensor . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 110
B. Master Switch: PIKK Family of Protein Kinases. .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 110
C. Adaptors and Mediators. . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 111
D. Effector Kinases: Chk1 and Chk2. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 113
E. From Components to Mechanisms . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 114
IV. Apoptotic Effectors in DNA Damage Response . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 115
A. Role of p53 in DNA Damage Response . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 115
B. p53-Related Proteins: p63 and p73 . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 117
C. Abl Tyrosine Kinase. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 118
D. Stress-Activated Protein Kinases: JNK and p38 . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 119
V. DNA Repair Proteins in Damage Signaling . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 120
A. Mismatch Repair Proteins. .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 120
B. UV-DDB Complex. . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 121
C. MRE11-RAD50 Complex . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 122
DNA Damage Responses . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 123
A. Integrative Surveillance. . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 123
B. Autonomous Pathways . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 125
VII. Future Prospects. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 127
References. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 128
I. Introduction
The continual DNA damage that occurs from physiological and
environmental agents is a threat to the integrity of the genome, viability
of the cell, and survival of the organism. Therefore, cells contain a variety
of DNA repair mechanisms (discussed in this book) for self-preservation
and self-protection. A large body of evidence has amply demonstrated the
importance of DNA repair to life. For example, certain base excision
repair and double-stranded-break repair functions are required for
viability in mice, whereas deficiencies in nucleotide excision repair or

102 WANG AND CHO
mismatch repair cause developmental defects and predispose mice and

men to cancer (Friedberg and Meira, 2003; see also relevant chapters in
this book). At steady states, DNA repair mechanisms are able to counter
continual damage, protecting an organism from the consequence of an
aberrant cell and protecting cells from the consequence of an aberrant
genome.
During the lifetime of a cell, circumstances may arise that cause excessive
DNA damage, calling for an increase in the capacity of repair. These are
the circumstances under which the cellular responses to DNA damage have
been studied. Experimentally, the investigation of DNA damage responses
(i.e., activation of DNA repair, inhibition of cell cycle progression, and
induction of apoptosis), has been conducted in eukaryotic cells under
conditions of genotoxic stress. The perturbations employed in these ex-
perimental protocols reveal how cells respond to an acute increase in the
level of DNA lesions. Therefore, DNA damage responses described in the
literature have mostly been observed following exposure to nonphysiologic
levels of ultraviolet (UV) radiation, ionizing radiation, oxidative stress, DNA
modifying chemicals, and drugs that inhibit DNA metabolic enzymes.
Interestingly, a number of these agents, including ionizing radiation,
DNA modifying chemicals (alkylating or crosslinking agents), and drugs
that inhibit topoisomerases or DNA polymerases have been applied empiri-
cally in cancer therapy with some—unpredictable—efficacy. Therefore,
studies on how cells respond to genotoxic stress are of relevance to cancer
therapy. Moreover, these stress responses are likely to reflect in part the
normal workings of the cell to preserve and protect the genome under
physiological levels of damage.
Our current knowledge of cellular responses to genotoxic stress is
largely derived from genetic studies in budding and fission yeast (Melo
and Toczyski, 2002; Nyberg et al., 2002). These studies have identified a
number of genes that are conserved in all eukaryotes to regulate cellular
responses to DNA damage (see below). A separate body of work on human
and mouse cell lines has identified additional genes not found in yeast,
most notably the tumor suppressor, p53, that play critical roles in DNA
damage responses. It is unclear whether all the players in the DNA damage
response network have been identified. Of the genes that have already
been identified, we know the functions of their products (see following).
However, we have not completely ascertained the rules that govern the
interplay among these gene products.
It is typically assumed that a ‘‘generic’’ program is launched on
genotoxic stress regardless of the type of lesion generated in the DNA.
This assumption is based on the observations in yeast cells that mutations
RESPONSES TO DNA DAMAGE 103
Table I
Conserved Core Components in DNA Damage Signal Transduction
Classification
Name Biochemical activity of putative role
Rad17-RFC Loading of the 9-1-1 clamp Damage sensor

(s.c. RAD24-RFC) onto damaged DNA
9-1-1 (s.p. Rad9-Hus1-Rad1) Heterotrimeric DNA clamp Damage sensor
(also known as RHR) May provide platform for
(s.c. DDC1, MEC1, RAD17) signaling complex
ATR, ATM, DNA-PK Protein kinase of the Master switch
(s.c. MEC1, TEL1; s.p. Rad3) PI3-kinase superfamily,
Phosphorylates target
substrates at SQ or TQ motifs
BRCA1, p53-BP1, NBS1, MDC1, Protein–protein interactions Adaptor/mediator
(s.c. RAD9; s.p. Crb2) for complex formation, and
subnuclear localization
Specific interaction domains
include the BRCT domain
and the FHA domain
Chk1 (also in s.c. and s.p.) Protein kinase Effector
Chk2 Protein kinase with Effector
(s.c. RAD53 FHA domain
and DUN1; s.p. Cds1)
s.c. for Saccharomyces cerivisiae; s.p. for Schizosaccharomyces pombe.
in genes such as RAD9 or MEC1 (see Table I) can affect responses to

a variety of genotoxic agents. In fact, many of these ‘‘general response’’
genes are conserved from yeast to man (see Table I), further support-
ing the notion of a ‘‘generic’’ program. Although these findings point to a
common core in the DNA damage response network, this common core is
but one component of the overall program. It should be noted that
different types of DNA lesions are recognized and repaired by different
proteins and enzymes. In addition, genotoxic agents can cause harm
to cellular components other than genomic DNA. Thus, the overall cellu-
lar responses to UV, ionizing radiation, or cisplatin are not neces-
sarily identical. Nevertheless, the cumulative data do support a common
theme in how cells react to genotoxic stress. The recurring theme appears
to be a temporally coordinated activation of DNA repair and cell cycle
checkpoints, followed by recovery, survival, or death. This chapter is
focused on a general discussion of how these temporal events may be
coordinated.
104 WANG AND CHO
II. Overview of Biological Responses to DNA Damage
A. General Framework of DNA Damage Responses

Because our current knowledge on DNA damage response has mostly
been gained from studies of individual gene products, this body of knowl-
edge has not permitted us to predict how a particular cell type would
respond to a specific type of DNA-damaging agent. The current knowl-
edge does indicate that the DNA damage-response network involves some
versatile players that can act on different stages with specified contexts
and cooperative strategies. Because it is not possible to deduce the
DNA damage response network with fine details at this time, we take the
approach of laying down a general framework that includes the various
responses (Fig. 1).
Construction of this framework is motivated by the consideration of two
overall biological objectives. The first objective of DNA damage response is
self-preservation, which is achieved by protecting the genome and allowing
for recovery of damaged cells. The second objective is self-protection,
which is achieved by eliminating damaged cells. In this framework, we
distinguish the various biological responses to DNA damage by their
reversibility. The transition of a biochemical reaction from a reversible
state to an irreversible state allows for the temporal regulation of the DNA
damage response (Fig. 1A).
B. Temporal Coordination of DNA Damage Responses

Temporally, we divide the biological responses to DNA damage into
three stages: the immediate early responses, which most likely occur
without the requirement for new gene expression; the early responses;
and the late responses, which are mostly irreversible (Fig. 1B).
1. Immediate-Early Responses
To protect the genome and the damaged cell, DNA repair is
activated and cell-cycle checkpoints initiated on DNA damage (Fig. 1B).
A DNA-damage signaling mechanism, composed of protein components
that are conserved from yeasts to mammals (see below), activates the
immediate early responses (Fig. 1B). The G2/M checkpoint, which
prevents the onset of mitosis, is installed through the phosphorylation/
inhibition of Cdc25C, a dual-specificity phosphatase that is required for
the activation of MPF (mitosis-promoting factor); that is, the kinase com-
plex of Cdc2/Cyclin B in mammalian cells (Fig. 2) (Zhou and Elledge,
RESPONSES TO DNA DAMAGE
Fig. 1. A general framework for the temporal coordination of DNA damage responses. (A) Biological responses to DNA damage
occur, temporally, from reversible to irreversible states. (B) Multiple outcomes are organized to depict immediate-early, early, and late
responses to DNA damage, as indicated by shaded boxes (see text for discussion).
105
106 WANG AND CHO
Fig. 2. Initiation and prolongation of cell-cycle checkpoints in response to

DNA damage. The G1/S and G2/M checkpoints are initiated by the phosphorylation
of dual-specificity phosphatases Cdc25A and Cdc25C to inactivate their functions.
Phosphorylation and inactivation of Cdc25A and Cdc25C are mediated by the Chk1
and/or Chk2 kinases, which are activated by DNA damage through the combined
actions of sensors, PIKKs, adaptors/mediators. The G1/S and G2/M arrest responses
can be prolonged by the accumulation of p21Cip1 or 14–3–3 sigma proteins, which
result from the transcriptional upregulation of these genes through p53, which is
stabilized and activated by the combined actions of the same proteins that initiate the
checkpoints.
2000). A parallel checkpoint, which prevents the onset of DNA replication,

is installed through the phosphorylation/degradation of Cdc25A, which
dephosphorylates/activates the mammalian Cdk2/Cyclin E (Fig. 2)
(Bartek and Lukas, 2003; Falck et al., 2002). Degradation of Cdc25A can
prevent S-phase entry and the continued progression through S-phase in
response to DNA damage (Bartek and Lukas, 2003).
2. Early Responses
Clonogenic survival occurs when damage is repaired and the cell cycle
checkpoints are lifted. If the damage is severe, either because of repair
defects or extreme destruction of the genome, death may occur within a
short period of time as a result of a general failure of metabolism. This
type of passive cell death may not be ‘‘regulated’’ by the damage response
network, and thus, it is not included in this framework. If repair is slow, the
ongoing repair causes the checkpoints to be prolonged. In mammalian
cells, the G1/S and G2/M arrest responses offer two examples for how we
distinguish between checkpoint initiation and prolongation.
The G1/S arrest can be initiated immediately after DNA damage by
biochemical mechanisms that block the firing of replication origins
(Bartek and Lukas, 2003) (Fig. 2). The G1/S arrest can be prolonged by
the up-regulation of p21Cip1, a heat-stable inhibitor of Cdk2/Cyclin-E
(Fig. 2). The accumulation of p21Cip1 protein is driven by an increase
in its mRNA, which is dependent on activation of p53 by DNA damage
(Fig. 2). Transcription and translation of p21Cip1 require more time;
therefore, we consider it to be an early response that prolongs rather than
initiates the G1/S arrest (Fig. 2).
The G2/M arrest can be initiated immediately after DNA damage by
biochemical mechanisms that block the onset of mitosis (Zhou and
Elledge, 2000) (Fig. 2). The G2/M arrest can be prolonged by the up-
regulation of 14–3–3-sigma, which is an adaptor protein that sequesters
Cdc2/Cyclin B in the cytoplasm (Chan et al., 1999) (Fig. 2). The accumu-
lation of 14–3–3-sigma protein is driven by an increase in its mRNA, which
is dependent on p53 (Fig. 2). Therefore, we consider the transcriptional
activation of 14–3–3-sigma to be an early response that prolongs rather
than initiates the G2/M arrest (Fig. 2).
3. Late Responses to DNA Damage

The late responses to DNA damage are irreversible. These irreversible
outcomes include clonogenic survival, adaptation, and death (mitotic
death, apoptotic death, mitotic catastrophe).
a. Clonogenic Survival vs. Mitotic Death/Premature Senescence The

prolongation of cell cycle checkpoints does not have to be permanent.
When damage is properly repaired, even with slow kinetics, the ‘‘check-
point prolongation’’ mechanism reserves the option of allowing resump-
tion of cell proliferation, leading to clonogenic survival. However, when
the prolongation of cell cycle checkpoints may become permanent, the
108 WANG AND CHO
damaged cell irreversibly withdraws from proliferation. This permanent

cell cycle arrest has been observed with fibroblasts exposed to high-dose
ionizing radiation and is customarily referred to in the radiation biology
literature as ‘‘mitotic death’’ (Hendry and West, 1997). Permanent with-
drawal of damaged cells from the cell division cycle has also been referred
to as ‘‘premature senescence’’ (Di Leonardo et al., 1994). Mechanistically,
the shutdown of cell cycle machinery in mitotic death or premature
senescence appears to involve the transcriptional repression of cell cycle
genes (e.g., cyclin-B and Cdc2) and requires the RB-family of transcription
corepressors (Badie et al., 2000; Taylor et al., 2001; Wang et al., 2001). It
thus appears that the permanent cell cycle arrest involves a program that is
triggered by DNA damage but that has a built-in temporal mechanism to
delay its execution.
b. Apoptosis vs. Necrosis Genotoxic stress can lead to the activation of

apoptosis, mediated by the release of proapoptotic factors from the mi-
tochondria (Lassus et al., 2002; Wang, 2001). Transduction of DNA dam-
age signal to mitochondria requires the tumor suppressor p53 (Fridman
and Lowe, 2003) (see following). Because p53-dependent apoptosis in-
volves the induction of transcription, the apoptotic response is delayed
relative to the onset of checkpoints. Activation of apoptosis eliminates
damaged cells and achieves the biological objective of protecting the
organism. Apoptotic cell death also protects the organism from the in-
flammation that can be triggered by necrotic cell death. Of course,
necrotic cell death resulting from metabolic failure in damaged cells also
protects the organism. Therefore, apoptosis is but one of many modes of
death that may result from genotoxic stress (Fig. 1B). The cellular decision
to commit suicide is highly dependent on the cell context. The temporal
regulation of apoptotic response to DNA damage is discussed further in
later sections of this chapter.
c. Adaptation Mitotic Catastrophe, and Genomic Instability In contrast

to ‘‘clonogenic survival,’’ which describes recovery of a damaged cell
with fully restored genome, ‘‘adaptation’’ describes the resumption of cell
cycle despite the persistence of DNA damage. The phenomenon of
adaptation to DNA damage has been observed in two yeast systems
(Sandell and Zakian, 1993; Toczyski et al., 1997). In one system, a galac-
tose-inducible endonuclease resulted in telomere loss; however, it was
found that yeast cells continued to divide despite the persistence of
damaged telomeres (Sandell and Zakian, 1993). In another system, an
irreparable double-stranded break was created in the budding yeast using
an inducible HO-endonuclease in a mutant RAD52 background (Toczyski
et al., 1997). The double-stranded break triggered the G2/M checkpoint,

but the break persisted because of the lack of RAD52 function. With time,
an adaptation to the persisting double-stranded break was observed as
yeast cells resumed mitosis despite this lesion (Toczyski et al., 1997).
Adaptation response to DNA damage can lead to catastrophic death
caused by mitotic failure (Fig. 1B). Adaptation response may also increase
the probability of necrotic cell death (Fig. 1B). If the adapted cells escape
mitotic catastrophe and resume cell proliferation, this would put
the genomes of daughter cells at risk. Thus, adaptation is a DNA damage
response that increases genomic instability and may precondition a cell for
tumorigenesis (Galgoczy and Toczyski, 2001). Further investigation of
adaptation to DNA damage in mammalian cells may yield important
insights on the mutability associated with malignant cancer cells.
C. Other Comments on the General Framework

We emphasize that the processes depicted in Figure 1 and discussed
above are more dynamic than could be presented in a two-dimensional
diagram. We would also emphasize that this framework is not a ‘‘molecular
pathway’’—none of the arrows represent a specific biochemical mecha-
nism. Rather, this framework temporally organizes the many cellular
responses that have been observed after cells incur DNA damage. In
particular, the framework highlights the importance of transitions from
reversible to irreversible events and how the status of DNA repair may
control these transitions. At the end of the chapter, we will discuss two
alternative models for how DNA repair may coordinate the checkpoint
and death responses to damage. Before those discussions, we will sum-
marize briefly the current knowledge on several key players in the DNA
damage response network.
III. Molecular Components for the Initiation of

DNA Damage Responses
As discussed above, the studies in yeasts have led to the identification of
genes that play critical roles in initiating DNA damage responses (Nyberg
et al., 2002). In the framework proposed in Figure 1, we would consider
these as the ‘‘immediate-early response’’ genes, which are best understood
at the molecular level. At present, the proteins and enzymes involved in
activating the immediate-early responses are thought to interact in a
‘‘signal transduction pathway’’ that relates the occurrence of DNA lesions
to repair proteins and to the cell-cycle engine. This signal transduction
110 WANG AND CHO
pathway has been reviewed extensively in recent years (Melo and Toczyski,
2002; Rouse and Jackson, 2002; Zhou and Elledge, 2000). Table I sum-
marizes some of these molecules required to initiate DNA damage re-
sponse, each of which is identified in yeast genetic studies. The molecular
functions of these yeast genes are conserved in mammalian cells (Table I).
A. DNA Damage Sensor

A hetero-trimeric DNA clamp known as the 9–1–1 complex is an impor-
tant ‘‘sensor’’ of damaged DNA. The 9–1–1 complex resembles the homo-
trimeric PCNA in structure and possibly also in function (Venclovas and
Thelen, 2000). The 9–1–1 complex derived its name from the fission yeast
genes Rad9, Rad1, and Hus1, each encoding a subunit of this heterotri-
mer, and this complex is conserved in mammalian cells (Caspari et al.,
2000; St Onge et al., 1999; Volkmer and Karnitz, 1999). The heterotrimeric
9–1–1 complex is loaded onto DNA at or near the damaged site by an
alternative RFC complex containing the Rad17 protein as its largest
subunit (Bermudez et al., 2003; Majka and Burgers, 2003). The biochemi-
cal mechanism of clamp loading by the Rad17-RFC is similar to the
loading of PCNA by the regular RFC at the origin of replication. The
Rad17-RFC is likely to recognize a structural cue resulting from the
processing of DNA lesions for loading the 9–1–1 clamp (Kai et al., 2001;
Zou et al., 2003). However, the precise biochemical mechanism for how
Rad17-RFC ‘‘senses’’ damaged DNA to load the 9–1–1 complex requires
further investigation. During DNA replication, the PCNA clamp functions
as a processivity factor for DNA polymerases. PCNA also provides a plat-
form for the recruitment of enzymes that complete the lagging strand
synthesis from Okazaki fragments (Hosfield et al., 1998). When loaded at
or near the damaged DNA site, the 9–1–1 clamp is thought to provide a
platform for the assembly of a damage-signaling complex (Osborn et al.,
2002; Zou et al., 2002). Whether the 9–1–1 clamp can also function as a
processivity factor of repair polymerases is still an open question.
B. Master Switch: PIKK Family of Protein Kinases

A family of protein kinases characterized by their large size (>200 kd)
and a highly conserved C-terminal kinase domain relating the phosphoti-
dylinositol-30 -kinase is found in all eukaryotic cells to regulate DNA
damage response (Hoekstra, 1997; Shiloh, 2003). These protein kinases
are commonly referred to as members of the PIKK family, which includes
the budding yeast MEC1 and TEL1, the fission yeast Rad3, the mammalian
ATR, ATM, and DNA-dependent protein kinase (DNA-PK). The PIKK
family members have been reviewed extensively in recent years because

they play essential roles in orchestrating cellular responses to DNA dam-
age (Shiloh, 2003). Moreover, the mammalian PIKK-family members are
involved in diseases; for example, ATM mutation in ataxia-telangiectasia,
and DNA-PK defect in severe combined immune deficiency (SCID)
(Hoekstra, 1997; Shiloh, 2003).
In mammalian cells, the ATM kinase is activated by ionizing radiation and
chromatin stress in vivo (Bakkenist and Kastan, 2003; Shiloh, 2003). The
mammalian ATR kinase can interact with and be activated by UV-damaged
DNA in vitro (Unsal-Kacmaz et al., 2002). A cofactor of ATR, ATRIP, can
interact with a single-stranded DNA/RPA complex (Zou and Elledge, 2003).
These observations support the conclusion of ATM and ATR kinases as
transducers of the DNA damage signal. The current research efforts on
the PIKK family members have been focused on the identification of their
protein substrates. The PIKK members phosphorylate serine or threonine
residues followed by a glutamine in the SQ or TQ motifs (Kastan and Lim,
2000). These simple motifs may not be sufficient to determine substrate
specificity; other protein–protein interactions may also be required for these
PIKK to select their substrates (Shiloh, 2003; Zou et al., 2002). To date, a
large number of proteins of diverse functions have been found to be
phosphorylated at SQ or TQ motifs following DNA damage in a PIKK-
dependent manner (Kastan and Lim, 2000; Shiloh, 2003). These include
DNA replication and repair proteins such as the MRE11-complex (see
below), transcription factors such as p53 in mammalian cells (see below),
and chromatin components such as histone H2AX (Burma et al., 2001) and
SMC1 (Kim et al., 2002). Importantly, PIKK members such as the yeast MEC1
and the mammalian ATM can phosphorylate proteins that are required for
the initiation of DNA damage response, including the adaptors (e.g., RAD9,
p53BP1), mediators (e.g., MDC1), and effector kinases (Chk1 and Chk2),
thus underscoring their roles as master switches.
C. Adaptors and Mediators

In mitogenic signal transduction, adaptor proteins play important roles
in the assembly of signaling complexes. Generally speaking, adaptor pro-
teins are composed of protein–protein interaction domains and function as
scaffolds to promote signal-dependent formation of specific protein com-
plexes. DNA damage appears to also induce the formation of protein
complexes, which involve scaffolding proteins that have been called ‘‘adap-
tors’’ or ‘‘mediators.’’ Two specific protein–protein interaction domains,
that is, the BRCT (breast cancer C-terminal) domain and the FHA (fork-
head associated) domain, have been found in several adaptors/mediators
112 WANG AND CHO
of DNA damage signal transduction (Bork et al., 1997; Durocher and

Jackson, 2002). The FHA domain preferentially binds peptides with a
phospho-threonine epitope (Durocher et al., 2000). The BRCT domain
can interact with another BRCT domain (Soulier and Lowndes, 1999) or
with phosphorylated serine or threonine residues (Manke et al., 2003;
Rodriguez et al., 2003; Yu et al., 2003). Of course, not all proteins with the
BRCT or the FHA domains are directly involved in the initiation of imme-
diate early responses to DNA damage (Bork et al., 1997). The FHA domain,
in particular, is widely distributed in nature and is found in prokaryotic
proteins that do not play any roles in DNA damage response (Durocher and
Jackson, 2002).
The budding yeast RAD9 gene product, which plays an essential role in
checkpoint activation, is a prototypical adaptor in DNA damage signal
transduction. The RAD9 protein contains a pair of BRCT domains at its C
terminus. In the budding yeast, DNA damage activates the phosphoryla-
tion of RAD9 through MEC1 and TEL1, and this phosphorylation event is
required for the activation of a downstream effector kinase RAD53
(Schwartz et al., 2002). Interestingly, there is not a mammalian protein
with contiguous sequence homology to the budding yeast RAD9. (The
mammalian Rad9 in the 9–1–1 complex is homologous to the fission yeast
Rad9 and the budding yeast DDC1.) In the fission yeast, Crb2 has a similar
function as RAD9. The fission yeast Crb2 protein contains a pair of BRCT
domains at its C terminus, and Crb2 is required for DNA damage to
activate a downstream effector kinase Chk1 (Mochida et al., 2004). A
number of BRCT-containing proteins are found in mammalian cells (Bork
et al., 1997). Among them, p53-BP1 (DiTullio et al., 2002; Wang et al.,
2002), MDC1 (also known as Kiaa0170) (Goldberg et al., 2003; Stewart
et al., 2003), BRCA1 (breast cancer associated 1) (Scully et al., 1997), and
NBS1 (Nijme gen breakage syndrome 1, or nibrin) (Lim et al., 2000) are
likely to play a RAD9-like role in transducing DNA damage signals. Each of
these mammalian BRCT proteins is phosphorylated in response to DNA
damage, and they contribute to the activation of DNA repair and cell-cycle
checkpoints. These mammalian BRCT-proteins are likely to have distinct
as well as redundant functions in DNA damage–signal transduction.
In yeasts, the FHA domain is found in the downstream effector kinases
RAD53 (budding yeast), DUN1 (budding yeast), and Cds1 (fission yeast),
and in Xrs2 of the MRE11/RAD50/Xrs2 complex. In mammalian cells, the
FHA domain is found in Chk2, in NBS1 of the MRE11/RAD50/NBS1
(MRN) complex, and in MDC1. The FHA domain of RAD53 interacts
with phosphorylated RAD9, and such interaction is required for RAD53
activation by DNA damage (Sun et al., 1998). The FHA domain of Chk2
mediates phosphorylation-dependent oligomerization and activation of its
kinase activity (Ahn et al., 2002; Xu et al., 2002). The FHA domain of NBS1
is required for relocalization of the MRN complex into nuclear foci in
damaged cells (Cerosaletti and Concannon, 2003). However, the FHA
domain of NBS1 appears to be dispensable for S-phase checkpoint
activation, an event that requires the MRN complex (see following).
The mammalian MDC1 and NBS1 proteins contain both the BRCT and
the FHA domains. MDC1 was named a ‘‘mediator’’ to distinguish it from the
BRCT-containing, RAD9-related ‘‘adaptors.’’ The MDC1 FHA domain and
BRCT domain appear to interact with distinct protein partners (Xu and
Stern, 2003); thus, MDC1 can function as a bridging protein to assemble
multiprotein megacomplexes. With NBS1, specific partners for its FHA
versus BRCT domains have not been described. Taken together, the current
evidence supports the idea that BRCT and FHA domains engage in the
formation of specific protein complexes in response to DNA damage.
D. Effector Kinases: Chk1 and Chk2

Members of the PIKK family are not the only protein kinases involved in
DNA damage response. Two other protein kinases, Chk1 and Chk2, are
also conserved through evolution and play important roles in regulating
DNA damage responses (Bartek and Lukas, 2003).
1. Chk1
The Chk1 kinase was discovered in the fission yeast as a protein
kinase that is activated by DNA damage to initiate the G2/M checkpoint
(Walworth et al., 1993). The classical G2/M checkpoint pathway activated
by DNA damage involves a Chk1-dependent phosphorylation of Cdc25
phosphatase, which is the universal activator of M-phase promoting factor
(Cdc2/Cyclin B) (Zhou and Elledge, 2000). Phosphorylation of Cdc25
prevents it from dephosphorylating Cdc2, thereby preventing the
activation of MPF (Zhou and Elledge, 2000). As a result, cells remain in
the G2 phase of the cell cycle, allowing time for DNA repair before the
onset of cell division. This Chk1-mediated G2/M checkpoint pathway is
conserved in metazoa (Bartek and Lukas, 2003; Melo and Toczyski, 2002;
Zhou and Elledge, 2000). Interestingly, the budding yeast Chk1 kinase
does not target Cdc25 but, rather, phosphorylates Pds1 to activate a
M-phase checkpoint in response to DNA damage (Liu et al., 2000b).
Whether the budding yeast Chk1-mediated M-phase checkpoint pathway
is also conserved in metazoan is currently unknown.
The knockout of Chk1 causes early embryonic lethality (Liu et al.,
2000a), indicating that the mammalian Chk1 kinase is required for viabili-
ty. This essential function of Chk1 may reflect a continual dependence of
114 WANG AND CHO
mammalian cells on the G2/M checkpoint pathway; however, it cannot be

ruled out that the mammalian Chk1 kinase may directly regulate cell
survival and that its essential function might be unrelated to the regulation
of Cdc25 phosphatase.
2. Chk2
The Chk2 kinase was identified in mammalian cells by virtue of its
homology to the fission yeast Cds1; hence, Chk2 is also known as the
hCds1 kinase (Bartek et al., 2001). The budding yeast RAD53 and DUN1
kinases are related to Cds1 and Chk1 in structure (Bartek et al., 2001). It
should be noted that although Chk1 and Chk2 belong to the protein
kinase superfamily, with the characteristic kinase domain consisting of an
ATP-binding lobe, a substrate binding lobe, and an activation loop, they
differ significantly outside of the kinase domain (Chen et al., 2000; Li et al.,
2002). The Chk2 subfamily of protein kinases contains the FHA domain,
with RAD53 containing two FHA domains (Bartek et al., 2001). As dis-
cussed above, one of the FHA domains of RAD53 interacts with phos-
phorylated RAD9 in response to DNA damage, and this interaction is
required for the phosphorylation and activation of RAD53 by MEC1
(Schwartz et al., 2002; Sun et al., 1998). In the budding yeast, RAD53 is
essential to the activation of DNA damage response (Melo and Toczyski,
2002; Zhou and Elledge, 2000). The fission yeast Cds1, by contrast, has
redundant function to that of Chk1, in that both kinases can phosphory-
late Cdc25 phosphatase (Melo and Toczyski, 2002). As discussed above,
the FHA domain of mammalian Chk2 also mediates phosphorylation-
dependent protein–protein interaction and plays a critical role in the
regulation of Chk2 kinase activity (Ahn et al., 2002; Xu et al., 2002).
Unlike Chk1, the mammalian Chk2 knockout does not cause cell
lethality, but compromises checkpoint and apoptotic responses to DNA
damage and predisposes to tumor formation (Hirao et al., 2002; Jack et al.,
2002; Takai et al., 2002). Interestingly, an inherited mutation of the CHK2
gene in humans is also associated with cancer predisposition (Bell et al.,
1999). Taken together, the current results indicate that Chk1 and Chk2,
although conserved through evolution, may be employed to serve redun-
dant or distinctive roles in DNA damage signal transduction, depending
on the organisms and cell types.
E. From Components to Mechanisms

Descriptors such as ‘‘sensors,’’ ‘‘master switches,’’ ‘‘adaptors/media-
tors,’’ and ‘‘effector kinases’’ suggest an understanding of the mechanism
by which DNA damage signal is transduced to regulate cellular processes.
However, it should be emphasized that such a mechanism is only implied,

rather than elucidated. The current understanding is based on genetic
studies, which provide insights on the hierarchical relationship among
these gene products but do not illuminate the biochemical nature of their
interactions. Therefore, the various descriptors should not be taken liter-
ally as components of a linear pathway. It is prudent to conclude at this
time that the mechanism of DNA damage–signaling transduction is not
known. A recent study has examined the subnuclear location and phos-
phorylation of some of the signaling components, including the NBS1 and
Chk2, during the first hour of genotoxic stress (Lukas et al., 2003). The
results confirm that these signal transducers are activated within minutes
of cellular exposure to genotoxic stress and describe the interesting spatial
segregation of damage sensors versus effector kinases in the nucleus
(Lukas et al., 2003). Exactly how these signaling molecules regulate the
late responses (i.e., adaptation or cell death) to DNA damage will await
further investigation.
IV. Apoptotic Effectors in DNA Damage Response

The apoptotic response to DNA damage has been extensively investi-
gated in mammalian cells because it involves the protein product encoded
by the p53 gene, one of the most frequently mutated genes in sporadic
human cancer. These studies have established that p53-dependent alter-
ation in gene expression is required for DNA damage to activate the
apoptosis machinery (Fridman and Lowe, 2003; Oren, 2003). Although
p53 function is necessary for the induction of apoptosis by DNA damage, it
is not sufficient. In addition, recent results indicate that p53 may activate
apoptosis through posttranscriptional mechanisms (Chipuk et al., 2004;
Mihara et al., 2003). The roles of p53 and several other signal transducers
in DNA damage-induced apoptosis are briefly discussed.
A. Role of p53 in DNA Damage Response

The p53 homo-tetramer is a transcription factor, with a defined DNA
binding domain, oligomerization domain, and transactivation domain
(Brooks and Gu, 2003; Oren, 2003). In response to DNA damage, p53 is
rapidly phosphorylated by the PIKK, Chk1, and Chk2 kinases (Brooks and
Gu, 2003; Giaccia and Kastan, 1998). Phosphorylation leads to the stabili-
zation of the p53 protein and activation of its transcriptional function. A
large number of p53-regulated genes have been identified, and they can
be loosely classified into four groups: DNA repair (e.g., Rnr), cell cycle
116 WANG AND CHO
(e.g., p21Cip1, cyclin G, 14–3–3-sigma), redox regulation (the PIG genes),

and apoptosis (Bax, Noxa, Puma) (Oren, 2003). Therefore, p53 is not a
‘‘dedicated’’ regulator of cell death; instead, p53 is a general regulator of
the transcriptional response to DNA damage.
The p53-dependent upregulation of p21Cip1, a heat-stable inhibitor of
cyclin-dependent kinase (Cyclin E/Cdk2, CyclinA/Cdk2), is responsible
for DNA damage–induced G1 arrest in mammalian cells. Up-regulation of
p21Cip1 may also contribute to DNA damage–induced premature senes-
cence (mitotic death). As discussed above, permanent withdrawal from the
cell cycle induced by high-dose ionizing radiation (IR) or other genotoxic
agents involves the transcription repression of Cdk and cyclins through
tumor suppressor RB and related p107, p130 ‘‘pocket proteins’’ (Badie
et al., 2000; Taylor et al., 2001; Wang et al., 2001). (The RB family of
proteins each contains several peptide-binding sites, that is, pockets, to
assemble transcription repression complexes at specific promoters; Chau
and Wang, 2003). The p21Cip1 protein can inhibit the phosphorylation of
RB, p107, and p130 by Cdk/cyclin; as a result, these pocket proteins are
able to assemble transcription repression complexes and thus suppress the
expression of cell cycle genes. Therefore, up-regulation of p21Cip1 not
only prolongs the G1 checkpoint but may also help to establish premature
senescence in collaboration with the RB family of transcription repressors.
The p53-dependent up-regulation of Bax, Noxa, and Puma contributes
to the induction of apoptosis because these proapoptotic Bc12 family
members can cause mitochondrial permeability transition to release sev-
eral apoptotic inducers including cytochrome c, AIF, Samc, and Omi
(Wang, 2001). Cells deficient in p53 are highly resistant to DNA damage–
induced apoptosis. However, p53-dependent up-regulation of Bax, Noxa,
and Puma could be observed in damaged cells that do not undergo
apoptosis (Takaoka et al., 2003). Thus, transcriptional activation of the
Bax, Noxa, or Puma gene expression is necessary but probably not suffi-
cient for DNA damage–induced apoptosis. Recent studies have suggested
that a fraction of the p53 protein exits the nucleus and binds to the
antiapoptotic Bc12 family members in the cytoplasm (Chipuk et al.,
2004; Mihara et al., 2003). This interaction is proposed to also be impor-
tant for altering the balance between the proapoptotic (Bax, Bak) and the
antiapoptotic (BclxL, Bcl-2) proteins, thus allowing the disruption of
mitochondrial integrity (Chipuk et al., 2004; Mihara et al., 2003).
The conclusion that p53 can either cause cell-cycle arrest or apoptosis in
response to DNA damage is without dispute. However, we have not
completely elucidated the rules that govern the selection of these two cell
fates (Oren, 2003). There is emerging evidence to indicate p53 can
selectively bind to different promoters, depending on the status of its
covalent modifications (phosphorylation and acetylation) (Brooks and

Gu, 2003; Giaccia and Kastan, 1998; Oren, 2003). In another view, the
choice between cell cycle arrest and apoptosis may not be made by p53
alone. Clearly, DNA damage signals activate other transcription factors
or posttranscriptional regulatory mechanisms, which may also contribute
to the decision between life without parole or death. This view is sup-
ported by the identification of proteins that are activated by DNA damage
to cause p53-independent apoptosis. A few of these proteins are discussed
below; however, we must emphasize that this discussion is by no means
comprehensive.
B. p53-Related Proteins: p63 and p73

In the mammalian genome, p53 is one of three related genes that
encode transcription factors with shared structures and functions. The
other two genes are p63 and p73, each encoding several protein products
through alternative promoter usage at the 50 end and alternative splicing
at the 30 end (Levrero et al., 2000; Melino et al., 2002). Genotoxic stress
induces the stabilization of p63 and p73 proteins and activates their
transcriptional functions (Flores et al., 2002; Gong et al., 1999). Current
evidence indicates that all three p53 family members contribute to DNA
damage–induced apoptosis. In fact, p63/p73 double-knockout mouse
cells are as defective as p53-knockout mouse cells in their apoptotic
response to doxorubicin or ionizing radiation (Flores et al., 2002). With
p63 or p73 single-knockout mouse cells, apoptosis to DNA damage is
reduced but not abolished, indicating that these two proteins may have
redundant functions in mediating apoptotic response to genotoxic stress
(Flores et al., 2002). Recent studies have identified genes that are com-
monly regulated by the three members of the p53 family as well as genes
that are uniquely regulated by p63 or p73 and not by p53 (Melino et al.,
2002). The transcriptional program that specifies the commitment to
apoptosis in damaged cells is therefore likely to be more complex than
previously thought.
Stabilization and activation of p73 by DNA damage require the Ab1
tyrosine kinase, which is activated by DNA damage (see following). The
Chk1 kinase also phosphorylates p73 to activate its function (Gonzalez
et al., 2003). At present, it is not known how DNA damage signals regulate
the p63 protein. The p63 and p73 genes, unlike p53, are seldom mutated
in sporadic human cancer (Melino et al., 2002). Because p73 can mediate a
p53-independent apoptotic response to DNA damage, the expression of
p73 in tumor cells is correlated with increased sensitivity to chemothera-
peutic agents that cause genotoxic stress (Irwin et al., 2003). Interestingly,
118 WANG AND CHO
p73 single-knockout cells also show reduced apoptotic response to tumor

necrosis factor-alpha (TNF-) (Chau et al., 2004). TNF-induced apoptosis
does not require new gene expression; on the contrary, actinomycin D or
cycloheximide, which inhibit transcription and translation, actually en-
hance the apoptotic response to TNF. Therefore, p73 may also activate the
apoptosis machinery through mechanisms other than the activation of
gene expression. The finding of p63/p73 and their role in apoptosis
regulation has informed us that studies of all three members of the p53
family are required to solve the mechanisms of DNA damage–induced
apoptosis.
C. Abl Tyrosine Kinase

The Abl protein is ubiquitously expressed in a variety of mammalian cell
types. This nonreceptor tyrosine kinase contains a number of functional
domains that endow it with cytoplasmic and nuclear functions (Wang,
2004). Abl contains nuclear localization and nuclear export signals and
distributes itself in both compartments in a dynamic equilibrium of import
and export. In the cytoplasm, Abl responds to signals from growth factors
and extracellular matrix to regulate F-actin (Woodring et al., 2003). In the
nucleus, Abl responds to signals from DNA damage and activates apoptosis
(Wang, 2000). Activation of nuclear Abl tyrosine kinase by ionizing radia-
tion requires a functional ATM kinase (Baskaran et al., 1997). Activation of
nuclear Abl tyrosine kinase by cisplatin requires the mismatch repair
protein, MLH1 (Gong et al., 1999; Nehme et al., 1999). Thus, Abl can be
considered a downstream effector kinase in DNA damage–signal transduc-
tion. Abl is not present in yeast cells. The Abl tyrosine kinase of lower
eukaryotes, such as Caenorhabditis elegans and Drosophila, does not appear to
have a nuclear function. Hence, participation of nuclear Abl in DNA
damage response may be limited to mammalian cells.
The activated nuclear Abl tyrosine kinase has been shown to phosphor-
ylate RNA polymerase II at its C-terminal repeated domain (Baskaran et al.,
1999), possibly to regulate gene expression following DNA damage. Nu-
clear Abl tyrosine kinase also phosphorylates p73 to stabilize the protein
and activate its apoptotic function (Costanzo et al., 2002). In myoblasts,
genotoxic stress activates nuclear Abl tyrosine kinase to phosphorylate and
inhibit MyoD function, resulting in a temporary halt of differentiation on
DNA damage (Puri et al., 2002). In addition, nuclear Abl tyrosine kinase
has been implicated in the tyrosine phosphorylation of DNA-dependent
protein kinase (DNA-PK), a PIKK family member involved in the nonho-
mologous end-joining repair of double-stranded breaks (Kharbanda et al.,
1997). Abl has also been shown to phosphorylate BRCA1 (Foray et al.,
2002), Rad51 (Yuan et al., 1998), Rad52 (Kitao and Yuan, 2002), and
UV-DDB2 (Cong et al., 2002). The effects of these tyrosine phosphoryla-
tion events on DNA repair, cell-cycle checkpoints, or cell death are not
understood.
Interestingly, activation of nuclear Abl tyrosine kinase by DNA damage is
dependent on cell adhesion (Truong et al., 2003). When fibroblasts are
detached from extracellular matrix (ECM) and then exposed to genotoxic
agents, nuclear Abl kinase is no longer activated (Truong et al., 2003). As a
result, detached fibroblasts are more resistant to DNA damage–induced
apoptosis than fibroblasts that are attached to ECM. This finding indicates
that Abl can integrate adhesion and DNA damage signals to regulate
apoptosis, and it implies that the delayed apoptotic response to DNA
damage may involve signal inputs other than DNA lesions.
D. Stress-Activated Protein Kinases: JNK and p38

The stress-activated proteins kinases (SAPKs) are classical MAP kinases
that are regulated by a kinase cascade, i.e., MAPKKK, MAPKK, and MAPK
(Morrison and Davis, 2003; Weston and Davis, 2002). A large body of
literature has accumulated on the kinase cascades that regulate SAPK
members such as JNK and p38; discussion of that literature is beyond
the scope of this chapter. It is sufficient to say that JNK and p38 are
activated by a variety of physiological signals as well as metabolic stresses,
including those that induce DNA damage (Weston and Davis, 2002). At
steady state, SAPK members are mostly localized to the cytoplasm. On
activation, SAPK members can translocate into the nucleus wherein they
phosphorylate transcription factors to regulate gene expression. The UV-
mediated activation of JNK involves cytoplasmic events that can occur in
enucleated cells, indicating that UV-induced DNA lesions are dispensable
in JNK activation (Rosette and Karin, 1996). Clearly, genotoxic agents can
damage other cellular components or generate reactive oxygen species to
activate SAPK members without input from DNA lesions. However, this
does not rule out the possibility that signals from DNA lesions might
directly activate a minor fraction of the SAPK members in the nucleus.
Activated SAPKs phosphorylate a number of transcription factors to
regulate gene expression (Weston and Davis, 2002). Some of the SAPK
members, notably p38, have also been shown to directly regulate cell
cycle progression by phosphorylating the Cdc25 phosphatase (Bulavin
et al., 2001). Other SAPK members, notably JNK, can directly
phosphorylate Bc12 family members to cause mitochondria-dependent
apoptosis (Lei and Davis, 2003). With mouse embryo fibroblasts (MEFs),
JNK1 and JNK2 are required for UV to induce apoptosis. MEFs derived from
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Jnk1/2-double-knockout embryos do not undergo apoptosis following UV

irradiation, despite the up-regulation of p53 (Tournier et al., 2000). The
requirement for JNK1/2 in the apoptotic response to UV and other geno-
toxic agents indicates that the damage of other cellular components or
oxidative stress may be important cofactors in determining the death
response to DNA damage.
V. DNA Repair Proteins in Damage Signaling

Given the notion that DNA damage signaling begins with the detection
of DNA lesions, it is not difficult to imagine the involvement of repair
proteins in this process because repair also begins with lesion detection. At
least three different lesion detection proteins, identified through the study
of DNA repair, have been linked to the regulation of DNA damage
responses. They are briefly discussed here.
A. Mismatch Repair Proteins

Mutations of mismatch repair proteins (i.e., MSH2, MSH6, MLH1,
or PMS2) contribute to microsatellite instability and increased mutation
rate in cancer cells. The MSH2/MSH6 heterodimer binds to mismatched
bases and several other types of DNA lesions (e.g., bases with bulky adducts
or platination; Bellacosa, 2001); thus, they can detect lesions. The MLH1/
PMS2 heterodimer binds to the MSH2/6 heterodimer. Analogous to the
bacterial mismatch repair mechanisms, MLH1/PMS2 are likely to partici-
pate in the recruitment of repair enzymes. Generally speaking, cells with
repair defects are more likely to die upon DNA damage. Therefore, it is
counterintuitive to find that MMR-deficient cells are more resistant to
DNA damage–induced apoptosis. Cancer cell lines or knockout mouse
cells deficient in MSH2, MSH6, MLH1, or PMS2 have each been shown
to exhibit defects in cell-cycle checkpoint or apoptotic responses, particu-
larly to chemotherapeutic agents such as temozolomide and cisplatin
(Bellacosa, 2001).
There are two general models for how MMR proteins regulate cell-cycle
checkpoints and apoptosis. The first is based on the concept of ‘‘futile
repair’’; that is, mismatch repair attempts to correct lesions that cannot be
repaired and that during this process generate double-stranded breaks,
which then trigger checkpoint and apoptosis (Li, 1999). The second is
based on the notion that mismatch repair proteins directly participate in
signaling. When encountering mismatched bases during DNA replication,
the MSH2/6-MLH1/PMS2 complex recruits repair enzymes to correct
such replication mistakes. It is conceivable that on encountering bul-

ky adducts or cross-linked bases, the MSH2/6-MLH1/PMS2 complex
adopts a different conformation that attracts signaling proteins to activate
checkpoints or apoptosis (Bellacosa, 2001).
Two lines of evidence have supported the second model (i.e., mismatch
repair proteins directly participate in DNA damage signaling). The first
line of evidence comes from the observed interactions among mismatch
repair proteins and components of the DNA damage–signaling pathway.
For example, MSH2/6 and MLH1 can associate with ATR and ATM
(Brown et al., 2003), two members of the mammalian PIKK family, and
PMS2 can associate with p73 (Shimodaira et al., 2003), an inducer of
apoptosis. The second line of evidence comes from the characterization
of MSH2 mutant protein; in particular, G674A (Lin et al., 2004). Cells
expressing this mutant MSH2 are defective in mismatch repair but profi-
cient in apoptotic response to cisplatin (Lin et al., 2004). This result
indicates that the repair function of MSH2 can be separated from its
signaling function. Although these two lines of evidence indicate mis-
match repair proteins to participate in DNA damage signaling, they
certainly have not ruled out a role for futile repair in the induction of
apoptosis.
B. UV-DDB Complex
UV irradiation induces cyclobutane pyrimidine dimers and (6–4)
pyrimidine–pyrimidone photoadducts in DNA, lesions that interfere with
transcription and DNA replication. The nucleotide excision repair ma-
chinery (NER), composed of several lesion-recognition proteins, helicases,
and nucleases, corrects UV-induced lesions. Among the components of
the NER machinery is a UV-damage DNA binding protein (DDB) complex
with two subunits: DDB1 and DDB2 (Keeney et al., 1993). Because DDB
directly binds to UV-induced lesions, it can function as a specific sensor of
UV damage. This sensor can recruit the NER machinery to facilitate repair
(Wakasugi et al., 2002). Alternatively, this sensor may recruit a signaling
complex to regulate the DNA damage response. Interestingly, cells derived
from Ddb2-knockout mice are resistant to UV-induced apoptosis, exhibit-
ing a delayed activation of caspase on UV-irradiation (Itoh et al., 2004).
This phenotype is similar to the resistance of mismatch repair – deficient
cells to cisplatin-induced apoptosis, as discussed above. At present, there
is no evidence to indicate that DDB contributes to futile repair of UV
lesions. Hence, DDB may not induce apoptosis through the creation of
double-stranded breaks at UV lesions. The alternative mechanism, that
DDB functions as a sensor of UV-induced lesions and directly participates
122 WANG AND CHO
in DNA damage signaling is implied, by the finding that Abl tyrosine

kinase associates with DDB and can phosphorylate DDB2 (Cong et al.,
2002). UV-induced apoptosis requires JNK activity as discussed above
(Tournier et al., 2000). The nuclear Abl tyrosine kinase has been suggested
to modulate JNK activity under conditions of genotoxic stress (Cross et al.,
2000). Interaction between DDB and Abl might therefore play a role
in controlling the apoptotic response to UV damage. A recent study has
found that DDB associates with Cu14A-Roc1, a cofactor in polyubiquitina-
tion of selected substrates (Groisman et al., 2003). Whether the DDB–
Cu14A complex participates in DNA damage signaling or the repair of
UV lesions by the NER is not known at this time.
C. MRE11-RAD50 Complex
The stable complex of MRE11 and RAD50 is found in prokaryotic and
eukaryotic cells. The crystal structure of the RAD50 subunit has revealed
a Zn-coordinated dimerization mechanism, which can join two molecules
of RAD50 with their globular N-terminal domains potentially separated
by 1200 Å (Hopfner et al., 2002). This structural design indicates that a
dimeric MRE11-RAD50 complex may bring together two DNA molecules
during nonhomologous end joining or homologous recombination
reactions, thus explaining its essential role in the repair of DNA ends.
Because the MRE11-RAD50 complex can recognize DNA ends, it can
function as a specific sensor of broken DNA in the genome (Petrini and
Stracker, 2003). Indeed, the MRE11 complex has been shown to be re-
quired for S-phase checkpoint activation in mammalian cells (Falck et al.,
2002; Petrini, 2000).
As discussed above, the mammalian MRE11-RAD50 complex (MRN)
contains a third subunit NBS1, which is encoded by the gene that is
mutated (although not completely lost) in the human genetic disorder
Nijmegen breakage syndrome (Williams et al., 2002). In mice, the knock-
out of Mre11, Rad50, or Nbs1 causes early embryonic lethality, and each
single knockout leads to the disappearance of the MRN complex (Luo
et al., 1999; Williams et al., 2002). In human, hypomorphic mutation of
MRE11 is associated with ATLD (Ataxia telangiectasia–like disease), and
that of NBS1 is associated with Nimejin Breakage Syndrome, with patho-
logical defects similar to Ataxia telangiectasia (Lee et al., 2003). As
discussed above, Nbs1 contains BRCT and FHA domains that mediate
the formation of protein complexes in response to double-stranded
breaks. Recent studies have suggested that an intact MRN complex is
required for ionizing radiation (IR) to activate the ATM kinase, one of
the master switches in orchestrating cellular responses to IR (Uziel et al.,
2003). Thus, the MRE11-RAD50-NBS1 complex appears to play multiple

roles, including lesion detection, DNA repair, and signal transduction.
The notion that lesion-binding proteins can function as ‘‘sensors’’ of
DNA damage to activate downstream signal transduction pathways is
gaining support but is far from proven. Further investigation is required
to directly link Mismatch Repair, UV-DDB, MRN, or other repair proteins
to the activation of checkpoints or apoptosis.

DNA Damage Responses
We propose two alternative mechanistic models for the temporal re-
gulation of cellular responses to DNA damage. We describe them as the
‘‘integrative surveillance’’ (IS) and the ‘‘autonomous pathway’’ (AP)
models (Fig. 3). These two hypothetical models represent two opposing
philosophical views, but they may not be mutually exclusive.
A. Integrative Surveillance
The IS model proposes the existence of a ‘‘regulatory hub’’ that actively
surveys the genome for lesions and for the status of repair to control the
cell cycle and the apoptosis machineries (Fig. 3a). The ‘‘regulatory hub’’
continuously monitors the extent of damage and the progress of repair,
integrates the information, and then inhibits cell cycle or activates apo-
ptosis. In the IS model, cell fate decisions are made logically based on the
rate of lesion accumulation/reduction. When lesions are increasing (i.e.,
the rate of lesion accumulation exceeds the rate of repair), the regulatory
hub activates repair and cell-cycle checkpoints. When lesions are decreas-
ing, (i.e., the rate of repair exceeds the rate of lesion accumulation), the
regulatory hub maintains checkpoints and inhibits apoptosis. When le-
sions persist (i.e., the levels do not reduce for an extended time), the
regulatory hub activates apoptosis.
The proteins and enzymes discussed above in this chapter can conceiv-
ably function in a ‘‘regulatory hub.’’ Protein complexes such as the 9-1-1-
heterotrimeric clamp, the MSH2/MSH6 heterodimer, the DDB hetero-
dimer, and the MRE11-complex can each function as a ‘‘sensor’’ of DNA
lesions; thus, they are able to monitor the levels of damage in the genome.
The PIKK master switch kinases can also directly or indirectly sense
damaged DNA. The Chk1 and Chk2 kinases, which are activated by
DNA lesions through the actions of damage sensors, PIKK members,
and adaptors/mediators, directly regulate the cell cycle machinery. The
PIKK members, Chk1 and Chk2, directly phosphorylate p53 to activate its
124 WANG AND CHO
Fig. 3. Alternative models for the temporal coordination of DNA damage responses.
(A) The ‘‘integrative surveillance’’ (IS) model proposes that signals indicating levels of
DNA damage and the progress of repair are integrated by a regulatory hub to either
activate or inhibit cell-cycle checkpoints versus apoptosis. The temporal coordination of
different biological outcomes is the result of deliberated decisions made by the
regulatory hub. (B) The ‘‘autonomous pathway’’ (AP) model proposes that DNA
apoptotic function. Therefore, it could be argued that these proteins

function in an integrative surveillance mechanism. The current data do
not explain how the damage sensors can monitor the ‘‘rate’’ of accumula-
tion or reduction in the levels of lesions. The integrative surveillance
model does not provide an explanation for adaptation, which is illogical
because adaptation allows a damaged cell to resume proliferation. Al-
though these questions remain, they have by no means ruled out the idea
of a ‘‘regulatory hub’’ in coordinating DNA damage responses.
B. Autonomous Pathways
The AP model proposes that the temporal regulation of cell-cycle
checkpoints, adaptation, apoptosis, and mitotic death is not achieved by
a logical ‘‘brainlike’’ mechanism but is the result of independent pathways
with built-in feedback mechanisms to cause delayed outcomes autono-
mously (Fig. 3b). In the AP model, DNA damage signal activates repair,
checkpoints, or cell death through distinct pathways, without the continu-
ous input from a ‘‘regulatory hub.’’ The DNA repair status affects the
checkpoint or the cell-death pathways solely by eliminating lesions and
therefore extinguishing the signals that trigger these pathways.
The current knowledge on DNA damage signaling is also compatible
with the AP model. The sensors, master switch, and effector kinases
respond to damaged DNA and transmit that information to the autono-
mous pathways. Together, these proteins send signals as long as lesions are
present in the genome (as illustrated by the gear wheel in Fig. 3b). The
signals are eliminated when lesions are removed by DNA repair. In the AP
model, cell-cycle checkpoints can be reversed through the built-in feed-
back mechanism while the gear wheel is still running, thus providing an
explanation for ‘‘adaptation’’ to DNA damage.
1. Autonomous Checkpoint Pathway
In the AP model, the checkpoint pathway has a time-dependent
negative feedback loop (Fig. 3b). Damaged DNA activates this pathway
to install the checkpoints. This pathway then proceeds through a series of
damage activates independent pathways with self-regulated feedback mechanisms.

There is not a central hub to coordinate the biological outcomes. A time-delayed
negative feedback loop in the checkpoint pathway can allow resumption of proliferation
despite persistent damage. By contrast, the cell death pathway is immediately
inactivated by a negative feedback loop. Only repeated activation of the death pathway
can eventually lead to cell death, either by apoptosis or mitotic death. The current data
can be accommodated by either model (see text for discussion).
126 WANG AND CHO
time-dependent steps, leading eventually to inactivation of the check-

points. Progression of this negative feedback loop can follow two possible
scenarios. In one, the feedback cycle can be set into motion by a DNA
damage signal and then run autonomously to the end. In this scenario,
cells recover from checkpoints at a predetermined time interval after
genotoxic stress, regardless of the extent of damage. If the DNA lesions
are eliminated within this predetermined time interval, cells recover and
undergo ‘‘clonogenic survival.’’ If the lesions are not completely elimi-
nated within this time period, cells undergo ‘‘adaptation’’ with damage
and are at risk for mitotic catastrophe, necrosis, or genome instability. In
an alternative scenario, progression through the negative feedback loop is
designed to require continual input of the DNA damage signal. If the
damage signal is eliminated, the feedback cycle is aborted, and cells
resume proliferation to undergo ‘‘clonogenic survival.’’ If DNA damage
signal persists long enough to allow completion of the entire feedback
cycle, cells undergo ‘‘adaptation.’’
2. Autonomous Death Pathway
In the AP model, cell death (either by apoptosis or through mitotic
death) is regulated by a series of feedback loops, which must be activated
multiple times to achieve death (Fig. 3b). The cell-death pathway is
activated immediately on DNA damage, but to no avail, because of a
built-in feedback inhibition (illustrated as the small negative feedback
loops in the diagram, Fig. 3b). At the completion of the first feedback
loop, the cell death pathway can be activated again if damage signal
persists. The repeated cycles of activation and inactivation can proceed
as long as the damage signal is present. The negative feedback mechanism
may be weakened through the successive rounds of activation. When the
feedback mechanism eventually fails, cell death occurs. The choice be-
tween apoptosis or mitotic death is determined by other factors relating to
the overall cellular context, but not through a ‘‘logical’’ evaluation of the
rate of lesion accumulation or the rate of DNA repair.
3. Negative Feedback Regulation of p53

DNA damage–induced death, either through mitotic death or apopto-
sis, requires p53 (Oren, 2003). The AP model would indicate p53 to be
regulated by an intrinsic negative feedback loop (Fig. 3b). Indeed, such a
feedback loop is well established, and it involves Mdm2, the inhibitor of
p53 (Michael and Oren, 2003). In nonstressed cells, p53 levels are kept
low through an Mdm2-mediated polyubiquitination and proteosome--
dependent degradation (Michael and Oren, 2003). Moreover, Mdm2
directly binds to p53 and inhibits its transactivation function (Michael
and Oren, 2003). DNA damage leads to the phosphorylation of p53 and
Mdm2, disrupting their interaction, allowing p53 to accumulate and to
activate the transcription of Mdm2 (Shiloh, 2003). This feedback loop has
been described at the level of single cells (Lahav et al., 2004). DNA damage
induces a wave of p53: its up-regulation occurs rapidly after ionizing
radiation, followed approximately 5–6 h later by its down-regulation
correlating with the up-regulation of Mdm2 (Lahav et al., 2004).
The single cell–based study has revealed a previously unknown property
of the p53 self-regulatory loop; that is, the peak height and the duration
of this p53 wave is not affected by the dose of ionizing radiation (Lahav
et al., 2004). Instead, IR dose affects the number of p53 waves a cell can
generate. A single p53 wave is observed following 2 Gy of IR, a dose that
rarely activates apoptosis or mitotic death in cultured mammalian cells.
Following 10 Gy of IR, a dose that activates apoptosis or mitotic death, two
or more waves of p53 are observed (Lahav et al., 2004). These observations
indicate that more than one wave of p53 activation, through the persis-
tence of DNA lesions, may be required to issue the cell death command.
The repeated activation of p53 may eventually wear down the autoinhibi-
tory loop to allow the accumulation of sufficient p53 to execute cell death.
Alternatively, the repeated activation of p53 may execute a multilayered
genetic program to trigger cell death without disrupting the autoinhibi-
tory loop. Future studies at the level of single cells will provide insights on
the temporal regulation of p53 and p53-dependent cell death response to
DNA damage.
VII. Future Prospects

Three objectives should propel the field of DNA damage response
forward. The first is to continue the identification of genes involved in
this process. The second is to increase our understanding of the biochem-
ical functions of the known gene products. The third is to elucidate the
mechanism of DNA damage signal transduction. These objectives can be
achieved with the conventional approaches already in practice. However,
new technologies and high-throughput methods will be required to pro-
vide a more coherent description of the DNA damage response program.
A systematic comparison of responses to different genotoxic agents in one
cell context will be helpful. These studies should include not only
biological recordings of cell viability, cell cycle profiles, and apoptotic
phenotypes but also molecular recordings of DNA lesions, the activity of
repair enzymes, protein kinases, and the extent of phosphorylation of
critical substrates. Examination of gene expression alteration as a function
of genotoxic dose and throughout a biologically relevant time course
128 WANG AND CHO
needs to be performed so that we can have a better understanding of the

entire protective program, not simply as a linear signal transduction
pathway but as a multilayered network. Many of these measurements
may have to be performed at the level of single cells, because recording
the averaged response of an entire population may miss important feed-
back loops that do not operate synchronously. Clearly, the field faces many
new challenges, which when met, will pay dividends that are worthy of the
investment.
Acknowledgments
We are supported by grants from the National Institutes of Health to J.Y.J.W. and a
postdoctoral fellowship from the Lady Tata Foundation to S.K.C. J.Y.J.W. is the Herbert Stern
Professor of Biology at the University of California, San Diego. We wish to thank members of
the Wang laboratory for critical comments, and La Jolla Scientific Management
(LaJollaSciManag@san.rr.com) for the graphic work.
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FUNCTIONS OF DNA POLYMERASES
By KATARZYNA BEBENEK AND THOMAS A. KUNKEL
Laboratory of Molecular Genetics and Laboratory of Structural Biology,

National Institute of Environmental Health Sciences,
Research Triangle Park, North Carolina, 27709
I. DNA Polymerase Families. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 137

II. Structures and Compositions of DNA Polymerases . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 139
III. Functions of DNA Polymerases . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 141
IV. Polymerases for DNA Repair . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 141
A. Polymerases for Nucleotide Excision Repair . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 143
B. Polymerases for Base Excision Repair . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 143
C. Polymerases for Interstand Cross-Link Repair . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 148
D. Polymerases for Nonhomologous End-Joining of
Double-Strand Breaks . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 149
V. Polymerases for Replicating Undamaged DNA. . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 150
VI. Polymerases for Sister Chromatid Cohesion . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 151
VII. Mitochondrial DNA Replication . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 152
VIII. Polymerases for Replicating Damaged DNA . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 152
IX. Polymerases and Cell-Cycle Checkpoints . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 155
X. Polymerases for Replication Restart and Homologous Recombination . . .. . . . . . 155
XI. Polymerases for DNA Mismatch Repair. . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 156
XII. Polymerases in the Development of the Immune System . . . . . . . . . . . . . . . . . . .. . . . . . 156
XIII. Biological Consequences of Polymerase Dysfunction. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 157
XIV. Closing Comments . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 158
References. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 159
I. DNA Polymerase Families

DNA polymerases are central players in DNA repair and replication, the
processes that duplicate genomes and maintain their integrity to ensure
faithful transmission of genetic information from one generation to the
next. Our appreciation for the enormous complexity of repair and repli-
cation processes has grown significantly in the past few years with the
discovery of a large number of DNA polymerases. Five polymerases are
now recognized in Escherichia coli, nine in Saccharomyces cerevisiae, and 16 in
humans (Table I) (Goodman, 2002; Hubscher et al., 2002; Shcherbakova
et al., 2003). Based on differences in the primary structure of their catalytic
subunits, DNA polymerases are classified into several distinct families
(Figs. 1 and 2). Family A is named after the E. coli polA gene that encodes
Pol I. Family A members also include the well-known bacteriophage T7
replicative polymerase and eukaryotic mitochondrial polymerase , as well
137
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PROTEIN CHEMISTRY, Vol. 69
138
Table I
DNA Polymerases in Escherichia coli, Saccharomyces cerivisiae, and Humans
Name Family Bacterial gene Human gene Yeast gene Mol. Wt. (kDa)a 30 Exo Other activities
Ec Pol I A pol A 103 þ 50 Exonuclease

(gamma) POLG MIP1 140 þ dRPlyase
(theta) POLQ 290 ATPase, helicase
(nu) POLN 100
Ec Pol II B polB 89 þ
(alpha) POLA POL1 (CDC17) 165 Primase
(delta) POLD1 POL3 (CDC3) 125 þ
" (epsilon) POLE POL2 225 þ
BEBENEK AND KUNKEL

(zeta) POLZ (REV3) REV3 353
Ec Pol III C dnaE 130 (separate
subunit)
(beta) X POLB 39 dRP lyase
AP lyase
(lambda) POLL POL4 (POLX) 66 dRP lyase, TdT
(mu) POLM 55 TdT
TdT TdT 56
(sigma) POLS (TRF4-1) TRF4 60
Ec Pol IV Y dinB 40
Ec PolV umuC 46
(eta) POLH RAD30 78
(RAD30A, XPV)
(iota) POLI (RAD30B) 80 dRP lyase
(kappa) POLK (DINB) 76
Rev1 REV1 REV1 138
a
Deduced from protein primary structure.
FUNCTIONS OF DNA POLYMERASES 139
as two newly identified polymerases in human cells, Pol (Seki et al., 2003;
Sharief et al., 1999) and Pol (Marini et al., 2003). Family B includes E. coli
Pol II, the product of the polB gene, and homologous such as the
replicative polymerases of bacteriophages T4 and RB69 and the eukaryotic
polymerases , , ", and . Family C includes the E. coli replicative poly-
merase Pol III, whose catalytic subunit is encoded by the polC (dnaE) gene,
and homologous polymerases present in most Gram-positive bacteria
(Bruck et al., 2003; Dervyn et al., 2001). Family D (not shown) contains
heterodimeric euryarchaea DNA polymerases (Pol II or PolD) (Cann and
Ishino, 1999). Family X includes the well-characterized mammalian Pol ;
more recently discovered eukaryotic polymerases , , and ; and a
template-independent polymerase, terminal transferase (TdT). Members
of the most recently named family Y include E. coli Pol IV and Pol V;
eukaryotic polymerases , and ; and a template-dependent deoxycytidyl
transferase, Rev1.
II. Structures and Compositions of DNA Polymerases

X-ray crystallographic studies (Beard and Wilson, 2003, and many re-
ferences therein) indicate that the catalytic subunits of polymerases in
different families share three common subdomains, often called the
fingers, palm, and thumb (Fig. 3; Ollis et al., 1985). These subdomains
form a cleft whose bottom is formed by the palm, which harbors three
catalytic residues (asterisks within red regions in Fig. 1) that coordinate
with two divalent metal ions. All DNA polymerases are believed to use a
common two-metal ion mechanism to catalyze the phosphoryl transfer
reaction for nucleotide addition (Steitz et al., 1994). Beyond these basic
features, polymerases are highly diverse, both between and even within
families. Their catalytic subunits range from relatively small proteins like
the 39-kDa human Pol to those as large as the 353-kDa human Pol
(Table I, Fig. 1). They interact with a variety of different accessory proteins
needed for repair or replication via noncatalytic domains and motifs, such
as the BRCT domain (the C-terminal domain of BRCA1, the product
encoded by the breast cancer susceptibility gene) or PCNA binding motifs
located in C-terminal regions of Pol and . Polymerases can also have
remarkably different polymerization properties. For example, the
efficiency with which different polymerases insert correct nucleotides
varies over an incredible 107-fold range (Beard et al., 2002), the number
of nucleotides they incorporate per binding event varies from one to
more than 10,000, and their fidelity varies by as much as 100,000-fold
(Table II; Kunkel, 2004). Polymerase substrate preferences also vary, from
preferential use of single-nucleotide gaps (Pol ) to preferential copying
140 BEBENEK AND KUNKEL
of damaged DNA (Pol ; McCulloch et al., 2004), to coordinated synthesis

of leading and lagging DNA strands by multiple polymerases acting as
integral components of a complex multiprotein replication machine
(McHenry, 2003). Some polymerases have additional enzymatic activities
such as 30 ! 50 exonuclease (yellow regions in Fig. 1), 50 ! 30 exonuclease,
50 -deoxyribose phosphate (dRP) lyase, ATPase (Pol ), or primase (Pol ).
These activities are located either in separate domains of the polymerase
polypeptide (e.g., yellow and gray domains in Fig. 3A–C) or reside in
separate but tightly associated subunits. Examples of the latter include the
30 ! 50 exonuclease activity in the subunit of E. coli Pol III and the
primase activity of Pol (Figs. 1 and 2).
III. Functions of DNA Polymerases

A complex network of DNA transactions must occur in cells to maintain
the appropriate balance between accurately maintaining genetic informa-
tion over many generations and permitting some diversity for the evolu-
tion of species, for the increased survival of microbes when subjected to
changing environments, and for the development of a normal immune
system. The physical and biochemical differences among polymerases
imply that each protein has evolved to fulfill specific roles in maintaining
this balance. In the following sections, we discuss the possible biological
roles of various polymerases, as suggested by their diverse properties.
IV. Polymerases for DNA Repair

We begin with repair transactions needed to provide clean substrates
for the replication fork. Normal cellular metabolism and exposure of cells
to exogenous genotoxicants produces DNA damage such as the loss or
modification of bases, single-strand and double-strand breaks in DNA, and
intra- and interstrand cross links. Multiple repair pathways exist to repair
DNA damage, and the specific repair pathway employed partly depends
on the type of damage experienced (Friedberg et al., 1995). A number of
Fig. 1. Modular organization of polymerases in different families. The modular

organization of the five Escherichia coli and 16 human polymerases is shown with the
number of amino acid residues in each polypeptide, as indicated. The polymerase
domains are colored red, and other domains or functional motifs are color coded as
indicated in the legend. A color gradient is used (see Pol III) to indicate that the
domain boundaries are not defined. (See Color Insert.)
these repair processes involve excision of damaged DNA followed by a

resynthesis step that requires a polymerase. However, the substrates for
DNA synthesis differ among the repair pathways, leading to different
polymerase requirements (Fig. 4).
A. Polymerases for Nucleotide Excision Repair

A variety of helix-distorting lesions, including ultraviolet (UV) radia-
tion–induced damage and bulky chemical adducts, are removed by nucle-
otide excision repair (NER), which can occur by distinct subpathways.
NER in prokaryotes and eukaryotes involves the same basic steps of
recognizing the damage, unwinding the DNA duplex containing the
damage, incision on both sides of the lesion to remove a damaged
oligomer, resynthesis of DNA to fill the gap, and ligation. In E. coli,
excision of the damage-containing oligonucleotide generates a 12–13-
nucleotide single-strand gap that is filled by Pol I. In eukaryotes, the gap
size is 30 nucleotides, and there is substantial evidence that the gap is
filled by one or both of the B-family polymerases, Pol or Pol ".
B. Polymerases for Base Excision Repair

When bases are lost by depurination or depyrimidination; when bases
are modified by alkylation, oxidation, or deamination; and when abnor-
mal bases (e.g., dUTP, 8-oxo-dGTP) are incorporated into DNA, the
resulting lesions are repaired by base excision repair (BER), which, like
NER, can occur via distinct subpathways (see Chapter 1; also see Beard and
Fig. 2. Subunit composition of polymerases in different families. The subunit

composition of E. coli and human polymerases is shown. The polymerase catalytic
subunits are depicted in red, and the accessory subunits are in other colors. The
abbreviations used are Kf, the large (Klenow) fragment of E. coli Pol I; T7,
bacteriophage T7 DNA polymerase, Klentaq, the large fragment of Thermus aquaticus
DNA polymerase I equivalent to Kf; Bs, Bacillus stearothermophilus DNA polymerase I
large fragment; RB69, replicative polymerase from bacteriophage RB69; Tgo,

Thermococcus gorgonarius DNA polymerase; 9 N-7, DNA polymerase from hyperthermo-

philic archaeon Thermococcus sp. 9 N-7; ASFV, African Swine Fever Virus family X DNA
polymerase; Dpo4, Sulfolobus solfataricus P2 DNA polymerase IV; Dbh, catalytically active
fragment of Sulfolobus solfataricus P1 DNA polymerase IV. 1Goldsby et al., 2001; 2Bemark
et al., 2000; Esposito et al., 2000; Wittschieben et al., 2000; 3Gu et al., 1994; 4Bertocci et al.,
2002; 5Bertocci et al., 2002; 2003; 6Gilfillan et al., 1993; 7McDonald et al., 2003;
8
Schenten et al., 2002. (See Color Insert.)
Fig. 3. Structures of DNA polymerases in different families. The images of T7 Pol,

Rb69 Pol, Pol , and Dpo4 were created based on their crystal structures in complex
with duplex DNA using coordinates from the Protein Data Bank under the accession
numbers 1T7P, 1IG9, 1BPY, and 1JX4 for T7 Pol, RB69 Pol, Pol , and Dpo4,
respectively. In the image of T7 Pol, RB69 Pol, and Dpo4, the subdomains palm, fingers,
and thumb of the polymerase domain are colored red, orange, and pink, respectively.
In Pol , the fingers are pink and the thumb is orange. The 30 exonuclease domains
(Exo 30 ) are colored yellow, and other domains are depicted in gray. The DNA template
strand (T) is navy blue, and the primer strand (P) is blue. The position of the 30
hydroxyl of the primer terminus is indicated by 30 . (See Color Insert.)
Table II
Error Rates of Polymerases in Different Families
Error rate 105

DNA Exonuclease
polymerase 30 ! 50 Family Substitution 1 deletions
Escherichia coli Yes C 0.6–1.2 0.025–1

Pol III
Escherichia coli Yes B 0.2 0.1
Pol II
Pol " Yes B 1 0.5
Pol Yes B 1 2
Kf(Pol I) Yes A 0.8 0.05
Pol Yes A 1 0.6
Pol No B 16 3
Pol No X 67 13
Pol No X 90 450
Pol No Y 580 180
Dpo4 No Y 650 230
Pol No Y 3500 240
Pol No Y 72,000 (TdGTP) —
22
(misinsertion at A)
Wilson, 2000; Bohr and Dianov, 1999; Lindahl et al., 1997). BER is initiated
by a DNA glycosylase, which recognizes the damaged base and removes it
by cleaving the N-glycosylic bond, leaving an apurinic/apyrimidinic (AP)
site. An AP site generated by a monofunctional DNA glycosylase (one
lacking an intrinsic AP lyase activity) is subsequently cleaved by an AP
endonuclease to create a nick with a 30 -OH and 50 -dRP terminus. Alterna-
tively, BER may be initiated by a bifunctional DNA glycosylase that also has
intrinsic AP lyase activity. After it cleaves the phosphodiester bond 30 of the
AP site, subsequent action of an AP endonuclease generates a single-
nucleotide gap with 30 -OH and 50 -phosphate termini. DNA-damaging
agents can also generate single-strand breaks that possess modified termi-
ni that need to be processed into substrates for polymerases or ligases
(reviewed in Caldecott, 2003a).
In E. coli, gap filling during BER is performed by Pol I. In mammals,
BER can involve different DNA polymerases. In the major mammalian
BER pathway, Pol inserts a single nucleotide onto the 30 -OH and then
removes the 50 -dRP group, using its dRP lyase activity. The resulting nick is
sealed by DNA ligase, completing the ‘‘short-patch’’ repair process. If the
dRP is modified or not cleaved by the dRP lyase activity of Pol , strand
displacement synthesis may generate a 2–13-nucleotide single-stranded
146
BEBENEK AND KUNKEL
Fig. 4. DNA polymerases involved in DNA repair and replication. See text for description. (See Color Insert.)
DNA flap that is removed by FEN1 flap endonuclease. Polymerases im-

plicated in synthesis during ‘‘long-patch’’ BER include Pol , Pol , and
Pol ". Which polymerase is actually used for any particular circumstance is
the subject of active investigations. S. cerevisiae lacks Pol , such that the
majority of BER in yeast may occur via the long-patch pathway, using Pol ",
Pol , and Pol (Wang et al., 1993). For further details on BER, see Beard
and Wilson (2000), Caldecott (2003b), Lindahl and Wood (1999), and
Chapter 1 of this book.
Like Pol , human Pol , Pol , and Pol also have a dRP lyase activity
(Table I), indicating that they too might participate in repair processes
that require removal of a dRP group. DNA Pol (Aoufouchi et al., 2000;
Garcia-Diaz et al., 2000) is a member of the X family. Pol and Pol have
similar domain organization and three-dimensional structures (Garcia-
Diaz et al., 2004), as well as several enzymatic properties in common. Like
Pol and other family X members, Pol lacks an intrinsic 30 ! 50
exonuclease activity, and it has low processivity when extending a primer
on a single-strand template. As for Pol , the processivity of Pol increases
when filling short gaps with a phosphate on the 50 end, and Pol can
substitute for Pol in reconstituted BER of uracil-containing DNA in vitro
(Garcia-Diaz et al., 2001, 2002). Thus, Pol is a likely candidate for BER
synthesis, perhaps being partially redundant with Pol or participating in
specialized BER reactions involving a subset of damaged bases, specific cell
or tissue types, or specific phases of the cell cycle. For example, the fact
that Pol has a high affinity for dNTPs is consistent with the hypothesis
that it might participate in BER or other repair processes under conditions
in which the nucleotide triphosphate pools are low (e.g., in quiescent
calls). In agreement with this, the expression of Pol is higher in cells
undergoing S to M phase transition and in quiescent cells.
As a member of the Y family, Pol has been implicated in the bypass of
lesions that block DNA replication (see following). In addition, some
properties of Pol are consistent with its possible role in specialized
BER processes. Pol has low processivity and can fill 1–5-nucleotide gaps,
and it can substitute for Pol to repair G–U and A–U pairs in DNA. In
considering which lesions Pol might possibly repair, it is notable that Pol
has a very unusual nucleotide incorporation specificity. Pol incorpo-
rates dTMP opposite template A much more efficiently than it forms the
three other correct Watson–Crick base pairs, and its insertion fidelity
opposite template A is relatively high (2 104), similar to that of Pol
. These observations led us to suggest that Pol could participate in BER
of uracil resulting from incorporation of dUMP during DNA replication
(Bebenek et al., 2001). Consistent with this idea is the fact that Pol
interacts with PCNA and colocalizes to replication foci (see Chapter 7).
Pol also has the unprecedented ability to misinsert dGMP opposite a

template T at a rate that exceeds that of correct dAMP incorporation.
Furthermore, on templates that contain two or more consecutive Ts,
preferential dGMP incorporation opposite the first T is followed by pref-
erential incorporation of A opposite the second template T. This remark-
able specificity led us to speculate that Pol may function in a specialized
BER reaction, replacing dGs that are inadvertently removed by a DNA
glycosylase from G–T or G–U mismatches that arise by deamination of
5-methyl-cytosine or cytosine (Bebenek et al., 2001).
The fourth human polymerase with dRP lyase activity is DNA polymer-
ase (Longley et al., 1998). As the only polymerase known to be present in
mitochondria, Pol is thought to be responsible for all DNA synthesis in
this organelle, including DNA replication and repair. Together with other
proteins in mitochondria implicated in BER, Pol may participate in
mitochondrial BER to remove damage resulting from reactive oxygen
species generated during oxidative phosphorylation.
C. Polymerases for Interstrand Cross-Link Repair

Interstrand cross links (ICLs) are highly cytotoxic lesions generated by
agents such as nitrogen mustard, psoralen, diepoxybutane, and cisplatin.
Repair of ICLs presents a special challenge, as both DNA strands are
damaged and neither strand retains the correct genetic information. In
E. coli, one repair pathway that has been well characterized (Van Houten,
1990) involves nucleotide excision repair, homologous recombination,
and the action of UvrD DNA helicase and Pol I. Another E. coli pathway
has been also described that depends on the function of NER and Pol II
(Berardini et al., 1999). The two pathways do not seem to be functionally
redundant, as cells deficient in either were hypersensitive to nitrogen
mustard; however, the exact function of each pathway is not yet deter-
mined. Repair of ICLs in mammalian cells is not yet well defined. On the
basis of studies showing that mutant alleles of some genes confer sensitivity
to cross-link-inducing agents in model organisms, polymerases implicated
in ICL repair include yeast Pol (see Chapter 6) and the Drosophila mus308
gene product. The latter has helicase motifs, and its C-terminal region
contains polymerase motifs with homology to E. coli Pol I. Mutations in the
mus308 gene cause hypersensitivity of cells to cross-linking agents and give
rise to chromosomal aberrations in treated mutant cells consistent with a
role of mus308 in repair of interstrand cross-links. Recently, two human
polymerases, Pol (Seki et al., 2003; Sharief et al., 1999) and Pol (Marini
et al., 2003), both members of family A, were identified by homology to the
Drosophila mus308 protein, which indicates their possible involvement in

ICL repair. Analysis of different mouse and human tissues indicated that
the expression of both genes is highest in testis. The recombinant proteins
showed polymerase activity on nicked double-strand DNA and on primed
single-strand DNA. Pol also has an intrinsic ATP-ase activity and helicase
motifs at the N terminus, although helicase activity has not been demon-
strated. On the basis of primer extension assays in the presence of a single
nucleotide, it was suggested that Pol incorporates G opposite template T
more readily than other family A polymerases.
D. Polymerases for Nonhomologous End-Joining of Double-Strand Breaks

During repair of double-strand breaks by nonhomologous end-joining
(NHEJ, reviewed in Critchlow and Jackson, 1998; Lieber, 1999), broken
DNA ends can be aligned using microhomology to create duplexes with
short gaps that need to be filled by a DNA polymerase. Filling of short gaps
during XRCC4-LigaseIV-dependent rejoining of double-strand breaks in
HeLa cell extracts requires Pol , including its N-terminal BRCT domain
(Lee et al., 2004). These and other observations (references in Lee et al.,
2004) suggest that, in addition to a likely role in BER, Pol also partici-
pates in NHEJ in human cells. Genetic (Leem et al., 1994; Wilson and
Lieber, 1999) and biochemical (Tseng and Tomkinson, 2002) studies
indicate that yeast Pol IV, the homolog of human Pol , is also involved
in NHEJ. Small gaps formed by the alignment of linear duplex DNA
molecules are preferential substrates for yeast Pol IV; the protein interacts
physically and functionally with Dn14/Lif1 complex, a core NHEJ factor;
and this interaction is mediated by the BRCT domain of Pol IV.
Another mammalian family X member, Pol , can extend primers
containing up to four mismatches (Zhang et al., 2001) and even perform
template-independent synthesis (Dominguez et al., 2000). Similar to Pol ,
Pol has a BRCT domain. It interacts with the Ku heterodimer, a major
NHEJ factor, and it stably associates with DNA in the presence of Ku and
XRCC4-ligaseIV. This complex can perform an end-joining reaction in-
volving annealing of partially overlapping DNA ends and the filling of a
single-nucleotide gap (Mahajan et al., 2002). Exposure of human cells to
ionizing radiation results in increased Pol levels, and Pol localizes in
nuclear foci containing double-strand breaks, indicating that it may also
be involved in a NHEJ pathway. Pol also has the remarkable ability to
efficiently incorporate ribonucleotides into DNA in vitro (Nick McElhinny
and Ramsden, 2003; Ruiz et al., 2003). The significance of this activity is
not yet well understood, but it has been suggested to permit activity under
conditions of low dNTP pools.
V. Polymerases for Replicating Undamaged DNA

Efficient removal of DNA damage by the repair pathways mentioned
above, combined with destruction of damaged dNTPs by enzymes (e.g.,
MutT, dUTPase) that sanitize dNTP pools (Ishibashi et al., 2003; Sekiguchi,
1996), provides undamaged substrates for the replication machinery.
During chromosomal replication, the two antiparallel DNA strands are
coordinately replicated (Kornberg and Baker, 1992; McHenry, 2003).
Because polymerases only synthesize DNA in the 50 to 30 direction, one
strand is replicated first as the leading strand, and the other is replicated
slightly later as the lagging strand. Leading-strand replication is largely
continuous, whereas lagging-strand synthesis is discontinuous and re-
quires multiple cycles of RNA priming and DNA synthesis to generate
Okazaki fragments, which are sealed after removal of the RNA. To per-
form efficient and accurate replication, replicative polymerases function
with multiple accessory proteins (Fig. 2).
In E. coli, the multisubunit Pol III holoenzyme is responsible for the
bulk of synthesis on both DNA strands during chromosomal replication,
whereas Pol I is involved in the processing of the Okazaki fragments. Pol
III comprises a core complex containing the catalytic subunit, the
subunit that has 30 ! 50 exonuclease activity for proofreading errors, and
the subunit that stabilizes the subunit (Fig. 2). Association of the core
polymerase with the clamp loader complex and the clamp forms the
highly processive and accurate holoenzyme that functions as an asymmet-
ric dimer for coordinated leading- and lagging-strand replication (Glover
and McHenry, 2001; Kelman and O’Donnell, 1995). In most Gram-positive
bacteria, two replicative polymerases are present, PolC and DnaE (Bruck
and O’Donnell, 2000). PolC and DnaE have high sequence similarity to the
subunit of E. coli Pol III. The subunit of PolC has an intrinsic 30 ! 50
proofreading exonuclease, whereas DnaE lacks an intrinsic exonuclease
and appears to have relatively low fidelity (Bruck et al., 2003). PolC and
DnaE function in a complex with the clamp and clamp loader, and it
has been suggested that PolC replicates the leading strand while DnaE
replicates the lagging strand (Bruck et al., 2003).
In eukaryotic cells, replication of undamaged chromosomal DNA in the
nucleus is performed by at least three DNA polymerases: Pol Pol , and
Pol ". The catalytic subunit of Pol is not highly processive and lacks
30 ! 50 exonuclease activity for proofreading errors, but it does have a
tightly associated primase activity for de novo synthesis of short RNA
primers at replication origins and for initiation of Okazaki fragments on
the lagging strand. Pol elongates these RNA primers to provide a short
DNA primer, and then a switch occurs to allow the bulk of chain
elongation by Pol or Pol ". Both Pol and Pol interact with the
eukaryotic sliding clamp PCNA, and both enzymes can synthesize DNA
processively. Both are accurate enzymes (Table II), because of the high
nucleotide selectivity of the polymerase active site and proofreading by
intrinsic 30 ! 50 exonuclease activities. Thus, both enzymes are well suited
for replicating large eukaryotic genomes. Yeast strains with a deletion of
the catalytic domain of Pol are inviable and are clearly defective in
replication. A strain with a deletion of the N-terminal region encoding
the polymerase activity of Pol is viable, albeit with severe growth and
replication defects, so long as the C-terminal region is expressed (Kesti
et al., 1999). Thus, another polymerase can partially substitute for Pol ".
However, a strain with a deletion of the C-terminal, noncatalytic domain of
Pol is inviable, indicating that this region is essential for some function
other than polymerization per se (Dua et al., 1999). The exact contribu-
tions of Pol and Pol to leading and lagging strand replication remain an
area of active investigation. The fact that they differ in primary structure
(Fig. 1) and protein partnerships (Fig. 2) implies that they have distinct
roles. Their functions may be differentiated for synthesis on opposite DNA
strands (e.g., Pol for the lagging strand and Pol for the leading strand,
or vice versa). Consistent with this hypothesis are data indicating that
30 ! 50 exonucleases associated with Pol and Pol can proofread
replication errors on opposite DNA strands during replication (Shcherba-
kova and Pavlov, 1996). Alternatively, or in addition, Pol and Pol
functions may be distinct for copying templates that differ by sequence,
timing in S phase, or chromosomal region (e.g., euchromatin versus
heterochromatin; see Fuss and Linn, 2002).
VI. Polymerases for Sister Chromatid Cohesion

To ensure accurate segregation of chromosomes to daughter cells
during mitosis, sister chromatids produced during replication are held
together by cohesion complexes until their separation in anaphase. The
product of the S. cerevisiae TRF4 gene, one of the proteins required for
establishing sister chromatid cohesion in S phase, has been reported to be
a family X DNA polymerase (Wang et al., 2000b). Originally designated as
Pol , it has since been designated Pol and is suggested to perform DNA
replication through the cohesion sites that could present an obstacle for
the replicative polymerases and ". Interestingly, it was recently reported
that the C-terminal domain of Pol interacts with Pol . This indicates that
Pol might be involved in coupling DNA replication and sister chromatid
cohesion. In addition to TRF4, eukaryotic genomes encode homologs of
TRF4 (e.g., TRF5 in S. cerevisiae and humans and Cid genes in fission yeast).
Cid1 was suggested to be a nucleotidyl transferase (Wang et al., 2000a),
and Cid13 has been demonstrated to have poly(A) polymerase activity
(Saitoh et al., 2002). In the latter study (Saitoh et al., 2002), the TRF4 gene
product was also shown to have poly(A) polymerase activity, leading to the
suggestion that an important function of these nucleotidyl transferases is
to polyadenylate mRNA.
VII. Mitochondrial DNA Replication

DNA polymerase is a member of family A and is the only cellular
polymerase known to be present in mitochondria (Kaguni, 2004). Thus, it
is believed to be responsible for replication of mitochondrial DNA, as well
as for any repair that occurs in mitochondria (e.g., BER; see above).
Human Pol is an accurate enzyme (Table II) and has an intrinsic
30 ! 50 proofreading exonuclease. It forms a tight complex with the p55
accessory subunit (Carrodeguas et al., 2001; Lim et al., 1999), which
increases DNA binding affinity, stimulates the polymerase and exonucle-
ase activities, and increases processivity.
VIII. Polymerases for Replicating Damaged DNA

DNA repair systems are not perfect and leave some lesions in DNA.
Moreover, some damage occurring during S phase may simply not be
repaired quickly enough to avoid an encounter with the replication
machinery. Many types of DNA damage, such as AP sites, UV photopro-
ducts, and adducts generated by polycyclic aromatic hydrocarbons distort
DNA helix geometry or alter base-coding potential. These lesions can stall
normal replication conducted by the major replicative polymerases, which
require correct base-pairing geometry for accurate and efficient replica-
tion (reviewed in Kunkel, 2004). To overcome the replication barrier
posed by such lesions, cells harbor multiple polymerases capable of
Translesion Synthesis (TLS). Most TLS polymerases belong to family Y,
members of which are found in organisms from bacteria to humans
(Ohmori et al., 2001). The properties of different family Y members are
described in Chapters 6–9. Here we present a brief overview.
Family Y polymerases typically (but not invariably) have relatively low
processivity, low catalytic efficiency, and low fidelity when copying undam-
aged DNA templates (Table II). The low fidelity reflects their lack of
30 exonucleolytic proofreading activity and also the intrinsically low
nucleotide selectivity of the polymerase active site. X-ray crystal structures
(Friedberg et al., 2001; Ling et al., 2001; Silvian et al., 2001; Trincao et al.,
2001; Zhou et al., 2001) indicate that the family Y polymerases may be able
to accommodate lesions because they have unusually small fingers and
thumb subdomains and because their active sites comprise smaller, un-
charged side chains. They may be more flexible and their active sites more
open and solvent accessible than polymerases in other families. In fact, the
active site of Sso Dpo4, a family Y polymerase, can simultaneously accom-
modate two undamaged template nucleotides (Ling et al., 2001), a cova-
lently linked cis-syn thymine–thymine dimer (Ling et al., 2003), or a bulky
benzo[a]pyrene diol epoxide adduct (Ling et al., 2004). Family Y TLS
polymerases can have very different properties, chief among them being
which lesions are or are not bypassed. Some of these differences may
depend on differences in an additional DNA binding domain distinct to
family Y enzymes, the little finger domain (Fig. 3), also called the wrist or
polymerase-associate domain. Data accumulated thus far indicate that,
depending on the DNA polymerase, the type of lesion, and the local
DNA sequence, translesion synthesis may either avoid or contribute to
mutagenesis.
Three E. coli polymerases are implicated in TLS, the family Y members
Pol IV (DinB) and Pol V (UmuD2C), and the family B member, Pol II (Fig.
4). All three are induced as part of the SOS response to environmental
stress, and all three modulate the ability of E. coli to survive during long
periods in stationary phase (Goodman, 2002). Recent studies in E. coli with
plasmids bearing different types of site-specific lesions show that all three
polymerases are involved in TLS and can modulate lesion-dependent
mutagenesis (Pagés and Fuchs, 2002). Human cells contain five TLS
polymerases: Pols , Pol , Pol , Pol , and REV1 (Fig. 4). Among these,
Pol is a member of family B and the others belong to the Y family. In
addition, DNA Pol has been shown to perform TLS synthesis in vitro
(Havener et al., 2003; Zhang et al., 2002), although there is as yet no
evidence that this ability is related to its in vivo function. Of these six
polymerases, only three are found in S. cerivisiae : Pol , Pol , and REV1.
Bypass of some lesions may be conducted by one polymerase that can
insert bases opposite the lesion and also extend the resulting primer
terminus (Fig. 5A). Other lesions may require two TLS polymerases for
bypass (Fig. 5B): one for insertion and another for extension, (e.g., Pol ;
see Chapter 6). Thus, translesion synthesis likely requires multiple
switches among polymerases and perhaps between polymerases and 30
exonucleases (e.g., intrinsic to Pol or Pol ") to allow proofreading of
errors introduced by the TLS pols (Matsuda et al., 2000), thus ensuring
efficient and accurate TLS. The mechanisms responsible for these enzy-
matic switches are under active investigation (e.g., see other chapters
and also McCulloch et al., 2004; Pham et al., 2001a). Coordination of
154
BEBENEK AND KUNKEL
Fig. 5. Models for DNA polymerase switching during translesion synthesis. (A) Model for lesion bypass by a single TLS
polymerase. (B) Model for lesion bypass by two TLS polymerases, wherein the first polymerase inserts a nucleotide opposite
the damaged site and the second extends the aberrant primer terminus. (See Color Insert.)
multiple polymerases during bypass likely involves the participation of

several polymerase accessory proteins. These include REV1, which inter-
acts with multiple TLS polymerases (Guo et al., 2003), and the E. coli
clamp and eukaryotic PCNA, which have been shown to interact with most
of the TLS polymerases (Fig. 3). In E. coli and S. cerevisiae, studies have
show that bypass synthesis depends on this polymerase-clamp interaction
(see Haracska et al., 2001; Pagés and Fuchs, 2002). Moreover, trafficking
among multiple polymerases may be modulated by posttranslational mod-
ifications of clamp proteins. For example, recent work indicates that the
protein–protein interactions of PCNA in replication and DNA repair can
be differentially modulated by distinct DNA damage–induced ubiquitina-
tion and sumoylation of PCNA (Hoege et al., 2002; Ulrich, 2004) and see
Chapter 10.
IX. Polymerases and Cell-Cycle Checkpoints

Replication stalled by an elongation barrier such as a blocking lesion
can initiate checkpoint responses in yeast and mammalian cells. Certain
checkpoint responses depend on Pol (Navas et al., 1995, 1996), specifi-
cally the noncatalytic C-terminal residues of Pol (Fig. 1) that encode a
putative zinc finger (Dua et al., 1998, 1999) and that interact with the
MDM2 protein (Vlatkovic et al., 2000), thereby enhancing polymerase
activity (Asahara et al., 2003). Defects in DNA damage checkpoints are
observed with mutants in the polymerase catalytic subunit (D’Urso et al.,
1995) and in the primase subunit of Pol (Marini et al., 1997), with a
mutant that inactivates the 30 exonuclease activity of Pol (Datta et al.,
2000), and with mutants in the fission yeast gene Cid1, a putative nucleo-
tidyltransferase in the TRF4/Pol family (Wang et al., 2000a). It is also
notable that one subunit of Pol shares significant homology and interacts
with MAD2, a key protein involved in the spindle assembly checkpoint
pathway (Murakumo et al., 2000). This checkpoint ensures that cells do
not enter mitosis and that chromosome segregation does not occur until
all chromosomes are properly attached to the mitotic spindle (reviewed
recently in Musacchio and Hardwick, 2002).
X. Polymerases for Replication Restart and

Homologous Recombination
When replication forks are stalled, they may be restarted by a ‘‘fork
regression’’ process, a rearrangement of DNA strands that allows
the complementary undamaged daughter strand to temporarily act as a
template for limited synthesis, followed by reestablishment of a normal

fork that ultimately results in accurate lesion bypass (see Fig. 5 in review by
Goodman, 2002). The DNA synthesis associated with this type of replica-
tion restart in E. coli is thought to be performed by Pol II (Pham et al.,
2001b), and in eukaryotes it may be a major replicative polymerase (e.g.,
Pol "; see discussion in Asahara et al., 2003). Alternatively, stalled replica-
tion may lead to double-strand breaks that can be repaired by homologous
recombination. The major replicative polymerases are likely to conduct
the DNA synthesis associated with homologous recombination (Holmes
and Haber, 1999; Jessberger et al., 1993). It has also been proposed that
Pol functions in replication-associated repair in early S phase and also in
replicating DNA in heterochromatin in late S phase (Fuss and Linn,
2002).
XI. Polymerases for DNA Mismatch Repair

Polymerization errors that escape proofreading can be corrected by
postreplicative DNA mismatch repair. This repair pathway recognizes
base–base and addition/deletion mismatches. Repair involves excision
of a region of the newly synthesized DNA strand containing the mismatch,
followed by accurate resynthesis of DNA. In E. coli, the enzyme responsible
for this DNA synthesis is Pol III. In human cells, resynthesis is performed
by a replicative polymerase (e.g., Pol ; Longley et al., 1997). In addition, it
has been suggested that exonucleases associated with Pol and Pol may
be involved in the excision step of the mismatch repair process. Mis-
matches can also result from DNA damage, such as G–U and G–T mis-
matches generated by deamination of cytosine and 5-methyl-cytosine,
respectively. In such cases, repair of the mismatch is initiated by a DNA
glycosylase, and the gap is filled by a BER polymerase such as Pol .
XII. Polymerases in the Development of the Immune System

The wide variety of immunoglobulins required for a full immune re-
sponse in humans and mice results from the combinatorial joining of
immunoglobulin gene (Ig) V, D, and J gene segments; from class switch
recombination; and from somatic hypermutation of variable (V) regions
(see Chapter 11). Several polymerases have been implicated in the DNA
synthesis required for development of a normal immune system.
Mammalian cells contain a template-independent family X polymerase
called terminal deoxynucleotidyl transferase (TdT). TdT contains a
BRCT domain characteristic of polymerases involved in DNA repair. It is

expressed in lymphoid tissue, and there it is involved in a specialized
polymerization reaction. In a template-independent manner, TdT inserts
nucleotides (so-called N-regions) at the junctions between the V, D, and J
elements during recombination to assemble expressed Ig heavy-chain
genes. This results in junctional diversity in the coding sequence, thereby
increasing the repertoire of immunoglobulins (Gellert, 2002; Neuberger
et al., 2003; Thompson, 1995). Pol is closely related to TdT, and it too is
highly expressed in lymphoid tissue. As mentioned above, it is thought to
be involved in NHEJ of double-strand DNA breaks. Studies with Pol /
mice indicate that Pol is involved in the processing of DNA ends during
Ig light-chain gene rearrangement at a stage when TdT is no longer
expressed (Bertocci et al., 2003).
The somatic hypermutation (SHM) process is characterized by the
frequent occurrence of base substitution mutations within a DNA seg-
ment of approximately one to two thousand bases of V regions in
Ig genes. These mutations are generated at a frequency that is perhaps
a million-fold higher than expected, given the very low rate of spontane-
ous mutation throughout the eukaryotic genome. The enzymatic mechan-
isms responsible for SHM are the subject of very active investigation (see
Chapter 11). The mutations are generated in two distinct phases that have
different base substitution specificity. SHM is likely to be initiated by
enzymatic cytosine deamination by the activation-induced cytosine deami-
nase (AID), followed by replicative-type or repair-type DNA synthesis.
Current biochemical and genetic evidence (reviewed in Chapter 11)
indicates that the polymerases responsible for this synthesis may include
members of family B, such as Pol , Pol , and Pol ", as well as members of
family Y, such as Pol or Pol .
XIII. Biological Consequences of Polymerase Dysfunction

There are now several examples in which mutations in polymerase genes
that inactivate or modify enzymatic functions have consequences for
human health (Kunkel, 2003). For example, several mutations in the
polymerase and exonuclease domains of human Pol have recently been
associated with progressive external ophthalmoplegia (PEO) (Copeland
and Longley, 2003; Ponamarev et al., 2002). PEO is a rare disease
characterized by muscle dysfunction resulting from the accumulation of
point mutations and large deletions in mitochondrial DNA that eventually
lead to loss of mitochondrial function. Humans carrying mutations in the
XPV (POLH) gene that inactivate the function of Pol suffer from
Xeroderma pigmentosum, a rare disease characterized by increased sus-

ceptibility to sunlight-induced skin cancer. Mice carrying a point mutation
that inactivates the 30 to 50 exonuclease of Pol and eliminates its proof-
reading function have a recessive mutator phenotype (Goldsby et al., 2002,
2001). The mice also have a recessive cancer phenotype characterized by
reduced lifespan (median survival, 10 months) and several tumor types,
predominantly of epithelial-cell origin. This implies that DNA polymerase
errors that are not proofread contribute to carcinogenesis. Thus, the
consequences of loss of proofreading during replication generally
conform to the mutator hypothesis for the origins of cancer, which posits
that an early event in tumorigenesis is the expression of a mutator
phenotype resulting from mutations in genes that normally function to
maintain genome stability (Loeb, 2001). These connections between poly-
merase dysfunction and disease indicate that it will be worthwhile in the
future to determine whether polymorphisms in DNA polymerase genes
are associated with adverse human health.
XIV. Closing Comments

A half century ago, in their seminal article describing the structure of
the DNA double helix, Watson and Crick (Watson and Crick, 1953) wrote,
‘‘It has not escaped our notice that the specific pairing we have postulated
immediately suggests a possible copying mechanism for the genetic mate-
rial.’’ Very soon thereafter, E. coli Pol I and human Pol were discovered
(reviewed in Kornberg and Baker, 1992). Fifty years later, we still do not
completely understand the functions of these two polymerases, so it is no
surprise that great uncertainty remains as to the precise functions of the
many other polymerases discovered since then, many of which were only
found in the last 5 years. What was certainly not appreciated 50 years ago
was the large number and amazing diversity of transactions involving DNA
synthesis required to faithfully replicate genomes that are diverse in
sequence, in functional composition, and in organization, and to stably
maintain them while they are being used for transcription and constantly
being insulted by normal cellular metabolism and by the external envi-
ronment. Although our knowledge of the existence and properties of
DNA polymerases has greatly expanded, there are many exciting questions
remaining to answer regarding the biological functions of each polymer-
ase and the mechanisms by which they are regulated so as to function in
the right place and at the right time. Many of the key issues, and our
current understanding of them, are addressed in the following chapters
and in the review articles liberally cited throughout this chapter.
Acknowledgments
We thank William Copeland, Matthew Longley, and Miguel Garcia-Diaz for critically
reading of this chapter and for offering thoughtful suggestions. We also thank Miguel Garcia-
Diaz for help in preparing the figues. TAK dedicates this chapter to the memory of Dale W.
Mosbaugh, an outstanding nucleic acid biochemist, a kind and generous human being, and a
very dear friend.
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CELLULAR FUNCTIONS OF DNA POLYMERASE z AND
REV1 PROTEIN
By CHRISTOPHER W. LAWRENCE

University of Rochester School of Medicine and Dentistry,
Rochester, New York, 14642
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 167
II. Enzymological Studies with Pol and Rev1p. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 172
A. Properties of Pol and Rev1p .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 172
B. In Vitro Studies of Pol and Rev1p on Lesion-Containing Templates . . . .. . . . . . 174
III. Genetic Analysis . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 178
IV. Processes Other than General Translesion Replication that Employ Pol
and Rev1p . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 182
A. Somatic Hypermutation. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 182
B. Double-Strand Break Repair. . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 184
V. Regulation of Pol and Rev1p and Interactions with Other Proteins . . . . . . . .. . . . . . 186
VI. Conclusions and Speculations . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 190
References . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 195
I. Introduction
DNA polymerase (Pol) and Rev1 protein (Rev1p) perform a variety of
important and, in some cases, essential, functions in eukaryotes, including
roles in DNA damage tolerance, in the development of diversity among
immunoglobulin genes, and in the repair of double-strand breaks by
homologous recombination. Originally discovered in budding yeast, Sac-
charomyces cerevisiae, the genes encoding these enzymes have been found in
all fully sequenced eukaryotic genomes, including those of other microbial
organisms, nematodes, mammals, and plants, suggesting that they are
present in all eukaryotes (Lawrence, 2002; Lawrence et al., 2000). Of the
various processes in which they participate, their roles in translesion
replication are probably the most widespread, though they may not be
universal. Like other DNA damage tolerance mechanisms, translesion
replication is concerned with overcoming the major consequence of un-
repaired damage in the genome; namely, its ability to block the progress of
replicases and thus prevent complete replication of the genome. Transle-
sion replication achieves this with the aid of Pol and Rev1p, together
with other specialized DNA polymerases, such as Pol. Although the
activities of Pol and Rev1p contribute only modestly to resistance to
DNA-damaging agents in yeast, they participate in processes that generate

168 LAWRENCE
the majority of spontaneous mutations and almost all of those induced

by overt mutagenic treatment. Interestingly, these enzymes appear to
contribute more substantially to damage resistance in at least some verte-
brates, raising the question of the molecular basis for this increased
importance. Even so, vertebrate Pol and Rev1p appear to be involved
in the production of most spontaneous and DNA damage–induced muta-
tions, like their yeast counterparts. Understanding the molecular process-
es underlying translesion replication and mutagenesis is therefore likely to
provide important insights into cancerogenesis and other genetic diseases
in which mutations figure prominently. In this context, it is also significant
that rev1 and rev3 yeast mutants are sensitive to many, though not all, of a
large set of anticancer agents (Simon et al., 2000).
The yeast REV3 gene, later shown to encode the catalytic subunit of
Pol, and the REV1 gene were first identified by Lemontt (1971) using a
screen for mutants with reduced frequencies of UV (ultraviolet)-induced
mutagenesis. REV7, encoding a second subunit of Pol, was subsequently
identified using a similar procedure (Lawrence et al., 1985a,b). The PSO1
locus, independently identified in a screen for mutants sensitive to
8-methoxy-psoralen plus 365 nm UV, was later found to be the REV3 gene
(Cassier-Chauvat and Moustacchi, 1988). Sequence analysis of cloned
REV3 (Morrison et al., 1989) revealed a predicted protein, with the motifs
of an enzyme belonging to the B family of DNA polymerases in the
classification of Ito and Braithwaite (Braithwaite and Ito, 1993; Ito and
Braithwaite, 1991), distantly related to DNA polymerase . Analysis of the
cloned REV1 gene (Larimer et al., 1989) identified a predicted protein
containing a 152-residue region with 25% identity and 42% similarity to a
segment of the E. coli UmuC protein, both of which were subsequently
designated as members of the recently designated Y family of DNA poly-
merases (Ohmori et al., 2001). Clones of the human homologs of REV3
(Gibbs et al., 1998; Lin et al., 1998; Morelli et al., 1998; Xiao et al., 1998),
REV1 (Gibbs et al., 2000; Lin et al., 1999) and REV7 (Murakumo et al.,
2000) have been isolated subsequently, as well as clones of mouse REV3
(Van Sloun et al., 1999) and mouse REV1 (Gibbs and Lawrence, unpub-
lished; Masuda et al., 2002). REV3 homologs have also been characterized
in Aspergillus nidulans (Han et al., 1998), Drosophila melanogaster (Eeken
et al., 2001), Neurospora crassa (Sakai et al., 2002), Arabidopsis thaliana
(Sakamoto et al., 2003), and chickens (Sonoda et al., 2003). The properties
of Neurospora REV1 and REV7 homologs (Sakai et al., 2003), and the REV1
homolog of chicken (Simpson and Sale, 2003), have also been investi-
gated. The domain structure of Rev1p, Rev3p, and Rev7p in budding
yeast, humans, and Arabidopsis thaliana is diagrammed in Fig. 1.
DNA POLYMERASE AND REV1 PROTEIN 169
Fig. 1. Domain structure and percentage identity of Rev1, Rev3, and Rev7 proteins
from yeast, humans, and Arabidopsis. A.t., Arabidopsis thaliana, S.c., Saccharomyces
cerevisiae, H.s., Homo sapiens. Numbers between lines connecting homologous domains
indicate percentage identity. Roman numerals I to VI above the Rev3 polymerase
domain indicate conserved sequence motifs.
Following the initial characterization of the yeast rev mutant pheno-

types, further investigations showed that they are deficient not only
for mutagenesis induced by exposure to 254 nm UV (Lawrence and
Christensen, 1976, 1978, 1979; Lawrence et al., 1984, 1985a; Lemontt,
1971, 1972) but also for mutagenesis induced by treatments with a wide
array of contrasting agents that also damage DNA. Rev3 and rev7 mutants
are deficient for mutagenesis induced by ionizing radiation (McKee and
Lawrence, 1979a,b), 4-nitroquinoline-1-oxide (Prakash, 1976), methyl
methane sulfonate and N-methyl-N 0 -nitro-N-nitrosoguanidine (Lawrence
et al., 1985b), ethyl methane sulfonate (Lemontt, 1972; Prakash, 1976),
8-methoxy psoralen plus 365 nm UV light (Henriques and Moustacchi,
1980), and different alkylating agents (Ruhland and Brendel, 1979).
170 LAWRENCE
Although fewer studies have been made with rev1 mutants, their pheno-
type appears to be similar (Lawrence and Christensen, 1976, 1978;
Lawrence et al., 1984; Lemontt, 1971, 1972; McKee and Lawrence,
1979a,b). Combined results from a variety of data suggest that
Pol function is required for the generation of 98%, and Rev1p func-
tion for 95%, of UV-induced base-pair substitutions. However, although
Pol function is required for the generation of 90% of UV-induced
frameshifts, Rev1p function is more variably involved in the production
of these events; for some, it is required to a similar extent as Pol, whereas
for others its involvement is much less (Lawrence and Christensen, 1978;
Lawrence et al., 1984).
As well as possessing a deficiency with respect to mutagenesis induced by
many DNA-damaging agents, rev1 and rev3 mutants of yeast are also anti-
mutators, with spontaneous mutation rates that are half to a quarter of
those seen in REV þ strains. Although this aspect of the rev3 mutant pheno-
type was first uncovered in a screen specifically designed to recover anti-
mutator mutants (Quah et al., 1980), and during the characterization of
pso1 mutants (Cassier et al., 1980), Pol activity was later found to be
responsible for the enhanced spontaneous mutagenesis observed in a
range of different genetic circumstances, which include the mutator phe-
notypes associated with rad1, rad6, rad18, and rad52 mutants (Roche et al.,
1994, 1995), with transcription (Datta and Jinks-Robertson, 1995) and
with double-strand break repair (Holbeck and Strathern, 1997). Rev1p, as
well as Pol, was found to be responsible for the increased spontaneous
mutation rate associated with overproduction of the 3-methyladenine DNA
glycosylase encoded by MAG1 (Glassner et al., 1998). Although the spon-
taneous mutations observed in the above investigations were principally
base-pair substitutions, Pol and Rev1p activities were also observed to be
largely responsible for the occurrence of spontaneous frameshift muta-
tions found in rad1, rad2, rad14, and rad52 strains and to be
entirely responsible for the complex events arising in these strains, in
which from one to five base-pair substitutions occurred in the region of
the þ1 insertion that led to the reversion of the lys2A746 allele employed
(Harfe and Jinks-Robertson, 2000). Pol was also responsible for the
enhanced frequencies of lys2BglII revertants found in stationary phase
cells of rad14 and rad16 strains (Heidenreich et al., 2004).
Investigations of REV gene function in organisms other than budding
yeast, although fewer in number, also support a role for these genes in
DNA damage–induced mutagenesis, and consequently in translesion rep-
lication. The organisms studied include filamentous fungi, plants, and
mammals, encouraging the conclusion that the properties observed are
likely to be found in almost all eukaryotes. Mutations in the UVSI gene of
Aspergillus nidulans, which encodes a REV3 homolog (Han et al., 1998), are
defective for UV mutagenesis (Chae and Kafer, 1993), and a similar
mutant phenotype has been observed for the upr-1 mutant of Neurospora
crassa, which encodes a REV3 homolog in this organism (Sakai et al., 2002).
REV1 and REV7 homologs have also been identified in Neurospora and were
shown to possess the same mutant phenotype (Sakai et al., 2003). Disrup-
tion of the REV3 homolog in A. thaliana led to increased sensitivity of
the plants to UV-B irradiation (>280 nm, peak 312 nm), -rays, methyl
methane sulfonate, and mitomycin C, though an influence on mutagene-
sis was not tested (Sakamoto et al., 2003). Reduction of cellular levels of
human Rev3p (Gibbs et al., 1998; Li et al., 2002) or Rev1p (Gibbs et al.,
2000) by high expression of antisense RNA decreased the frequencies of
6-thioguanine resistant mutants induced in human fibroblasts by 254 nm
UV by about sevenfold and more than 20-fold, respectively. High expres-
sion of REV3 antisense RNA also reduced the frequency of 6-thioguanine-
resistant mutants induced by benzo[a]pyrene diol epoxide by four- to
sixfold in these cells (Li et al., 2002). In a related approach, reduction
of Rev1p expression with a ribozyme construct decreased the frequency of
254-nm UV-induced 6-thioguanine-resistant mutants in human cells by
two- to threefold (Clark et al., 2003). Last, the frequency of spontaneous
6-thioguanine-resistant mutants was decreased several fold by high levels of
REV3 antisense RNA in an msh6 human fibroblast cell line (X. Li and V.M.
Maher, unpublished data, cited in Lawrence et al., 2000). Results from
each of these investigations therefore resemble, at least qualitatively, those
observed with yeast, and indicate that the functions of Pol and Rev1p are
conserved from fungi to plants and humans. However, the involvement of
Pol in translesion replication may not be universal. Although Drosophila
melanogaster possesses a REV3 homolog, no evidence could be found in
this organism for its involvement in forward mutagenesis induced by
x-rays, 4-nitroquinoline-1-oxide, or methyl methane sulfonate; instead, it
appears to be concerned with repair (Eeken et al., 2001).
This chapter reviews information about the properties and functions of
Pol and Rev1p in the diverse processes within which they play a part.
Although most of these data come from yeast, important results have also
been obtained from a variety of other species, including the mouse, hu-
mans, and chickens. A combination of investigations over the last several
years, examining the enzymology, genetics, and cell biology of Pol and
Rev1p, has done much to advance our understanding of their cellular
functions, but conflicting interpretations remain with respect to several
important issues. A second aim of this chapter is, therefore, to review the
different models proposed for Pol and Rev1p function, and examine
the data that are used to support them.
172 LAWRENCE
II. Enzymological Studies With Pol And Rev1p

In vitro studies of the purified yeast enzymes and, for Rev1p, its mam-
malian counterpart have established some of the basic properties of
these proteins. They have also been used to investigate the role of these
enzymes, often in combination with other DNA polymerases, in replica-
tion past various lesions, leading to proposals concerning the in vivo
function of the enzymes. It is likely, however, that such reactions lack
important factors present within cells, emphasizing the need for validation
of the models with in vivo studies, as discussed in Section III.
A. Properties of Pol z and Rev1p

Although yeast REV3 encoded a predicted protein that contained motifs
characteristic of a DNA polymerase, biochemical demonstration of this
activity was obtained only after identification of a second subunit, encoded
by REV7 (Nelson et al., 1996a; Torpey et al., 1994). All in vitro studies of
Pol have employed this two-subunit enzyme from yeast; efforts to produce
it from mammalian genes, or those from other organisms, have so far been
unsuccessful. Because native Pol has not been isolated from any species,
probably the consequence of very low cellular levels, it is uncertain whether
other subunits exist, or whether the two-subunit enzyme possesses a fully
normal activity. In particular, a variety of evidence suggests that Rev1p is
essential for Pol activity, but a functional association with this protein has
not yet been reconstructed, perhaps because of a requirement for other
proteins.
As expected for a B-family polymerase, the activity of the two-subunit
enzyme is DNA-template dependent and employs all four dNTPs. It is,
however, poorly processive, adding three or fewer nucleotides per binding
event, and it lacks a 30 –50 proofreading exonuclease activity (Nelson et al.,
1996a). Compared to other B-family DNA polymerases, Pol has an un-
usual facility for extending primers with terminal mismatches ( Johnson
et al., 2000; Lawrence and Hinkle, 1996; Lawrence et al., 2000), suggesting
a general capability for elongation from structurally abnormal termini of
the kind presented by DNA lesions. Apparent extension efficiencies from
terminal mismatches, as determined by the method of Mendelman et al.
(1990), were strongly sequence dependent, but were always greater for
Pol than for Pol, which, like Pol, also lacks proofreading activity. In one
sequence context, Pol was twofold to 1265-fold more efficient than Pol
within the complete set of mismatches examined, and in another, Pol was
fivefold to 1,226-fold more efficient. For some mismatches, extension by
Pol was only a few fold less efficient than extension from the correctly
paired terminus. In the most extreme case, in which a primer terminal G

was mispaired with template T, apparent extension efficiencies using Pol
were 54% and 35% of the values for the AT match in the two sequence
contexts examined, whereas for Pol, the corresponding efficiencies were
1.7% and 0.33% (Lawrence et al., 2000).
In addition to an unusual facility for extension from mismatched ends
of primers, Pol appears to incorporate nucleotides with only a moderate
fidelity. On lesion-free templates, Pol was found to have an incorporation
error rate of between 4 103 and 2 105, depending on the particular
template base and incoming nucleotide examined, with most values lying
between 104 and 105 ( Johnson et al., 2000). Although the authors
suggest that these values approach those for Pol, citing data from Thomas
et al. (1991), results in the latter paper indicate that the error rates for Pol
are at least 10-fold lower, with values of between 2.9 105 and 3.6 106,
depending on the site studied. However, because the data from Thomas
and coworkers were calculated from LacZ forward mutation rates during
gap-filling synthesis on an M13mp2 template, a very different procedure to
that used by Johnson and coworkers, it would be useful to directly com-
pare the two enzymes using the same primed templates and methodology.
On the basis of these data, it has been suggested that Pol is incapable of
inserting nucleotides opposite lesions, but extends termini resulting from
insertions by other enzymes ( Johnson et al., 2000; Prakash and Prakash,
2002), a model that is discussed in Section VI.
Yeast Rev1p has two distinct functions, neither of which is fully under-
stood. The most general, with respect to different DNA lesions, is its
apparent requirement for Pol activity (see Section III), and a possible
mechanistic model for this function is given in Section V. The second,
more specialized, function is a DNA template-dependent deoxycytidyl
transferase activity. In the original qualitative study of this activity, Rev1p
was found to preferentially insert dCMP opposite a template abasic site
and, less efficiently, opposite template guanine and adenine nucleotides
(Nelson et al., 1996b). The reaction was highly specific for dCMP and
could not use ribonucleoside triphosphates. This finding explained a
prior observation (Gibbs and Lawrence, 1995), that dCMP was inserted
in 60%–85% of the bypass events at a site-specific abasic residue in vivo, a
preference that was later shown to be entirely dependent on Rev1p
(Nelson et al., 2000). Similar to yeast Rev1p, both the human (Lin et al.,
1999; Zhang et al., 2002) and the mouse enzyme (Masuda et al., 2002)
possess a deoxycytidyl transferase activity, though low levels of dGMP and
dTMP incorporation were also observed with these enzymes. Steady-state
kinetic studies with each of these three Rev1p (Haracska et al., 2002;
Masuda et al., 2002; Zhang et al., 2002) indicated that dCMP insertion
174 LAWRENCE
was most efficient opposite sites of template guanine, though insertion

at an abasic site was more efficient than at template A, T, or C, and
the degree of preference for template guanine varied between studies.
Because the efficiency of dCMP insertion by mouse Rev1p was influenced
strongly by sequence context, the quantitatively disparate results may arise
from this variable, though there may also be intrinsic differences between
the proteins from each organism.
The extent of the preference for dCMP incorporation at template gua-
nine rather than an abasic site was particularly great in results with the
yeast enzyme, leading the authors to propose that Rev1p is a G-template-
specific DNA polymerase (Haracska et al., 2002). Because there is clear
evidence for Rev1p-dependent insertion of dCMP opposite an abasic site
in vivo in yeast (Gibbs and Lawrence, 1995; Nelson et al., 2000; Otsuka et al.,
2002a; 2004), such a designation appears inappropriate. Moreover, it seems
unlikely that eukaryotes require an inefficient, highly unprocessive, Y-family
enzyme to incorporate dCMP opposite normal guanine residues in the
presence of highly efficient and accurate enzymes such as Pol.
The apparent conflict between the in vivo and in vitro results may
perhaps be explained by Rev1p existing within a complex in the cell, in
which dCMP insertion is inhibited at template sites other than those with
abasic residues and, perhaps, some other lesions. Finally, the deoxycytidyl
transferase activity, even if not essential for the bypass of abasic sites,
nevertheless doubles the efficiency of replication past this lesion in yeast
(Otsuka et al., 2002b; see also Section III), perhaps explaining why the
activity has been maintained throughout evolution.
B. In Vitro Studies of Polz and Rev1p on Lesion-Containing Templates

A variety of investigations has been carried out to examine the roles of
Pol and Rev1p on lesion-containing templates, usually in combination with
other DNA polymerases or proteins concerned with translesion replication.
Oligonucleotide templates containing a diverse variety of lesions have
been used in such studies, including those carrying an abasic site,
thymine–thymine pyrimidine (6–4) pyrimidinone adduct [T-T (6–4) photo-
adduct], thymine–thymine cis-syn cyclobutane dimer (T-T dimer), acetyl
aminofluorene-guanine adduct (AAF-G), (þ) and () anti-benzo[a]pyrene
diol epoxide (BPDE), 7,8-dihydro-8-oxoguanine (8-oxoG), O6-methyl gua-
nine (6MeG), thymine glycol, and acrolein derivatives of guanine. Pol
appears to play a part in the bypass of all of these lesions, apart from the
T-T dimer, though the particular role may vary.
Abasic residues are one of the most abundant types of DNA damage,
with 10,000 of these lesions arising daily in mammalian genomes
(Lindahl, 1993). At the same time, they constitute a fairly severe block
to continued replication. The identity of the enzymes used by yeast to
replicate past an abasic site has been investigated by several groups. Nelson
et al. (1996b) showed that although Pol alone replicated past an abasic
residue very inefficiently, it readily extended the primer resulting from
incorporation of dCMP opposite the lesion by Rev1p. Yeast Pol, however,
was unable to extend from such an insertion. This result is consistent with
in vivo data showing dependence of such bypass on Pol and Rev1p, and
preferential incorporation of dCMP opposite the abasic site (Gibbs and
Lawrence, 1995; Nelson et al., 2000). Yuan et al. (2000). On the other
hand, examined insertion opposite the lesion by Pol, which was unable to
further extend the primer, followed by elongation with Pol. Pol princi-
pally inserted dAMP and dGMP opposite the abasic residue, though less
frequently it incorporated the other two nucleotides. In contrast, a steady-
state kinetic analysis (Haracska et al., 2001a) indicated that insertion
opposite this lesion by either yeast or human Pol was very inefficient.
This was partly alleviated by the presence of PCNA, RPA, and RFC
(Haracska et al., 2001a), but Pol could not extend from these insertions.
Yuan and coworkers (2000) also presented data indicating that yeast Pol
was capable of bypassing an abasic site, principally incorporating dAMP,
but such bypass was achieved only with high enzyme-to-template ratios that
are probably uncharacteristic of in vivo conditions. The identity of the
enzymes responsible for replication past abasic residues was also investi-
gated by measuring bypass product yields resulting from in vitro reactions
containing combinations of Pol, with either Pol or Rev1p (Haracska et al.,
2001c). None of these enzymes alone was found to be capable of replicat-
ing past the abasic residue, though, under the conditions used, Pol could
insert dAMP opposite the lesion, and Rev1p, as expected, could insert
dCMP, with Pol capable of extending from both of these nucleotides. Of
these two combinations, it was concluded that bypass of this lesion in vivo
entailed insertion opposite the abasic site by Pol, rather than by Rev1p,
followed by extension of the primer by Pol, because the reaction efficien-
cy was greater. In a running-start assay, where the primer was set back 15
nucleotides from the abasic site, the amount of bypass product yielded by
the Pol/Rev1p combination was 33% of that produced by Pol/Pol,
whereas in a standing-start assay, where the primer abutted the lesion, the
relative yield was 55%. Of these two estimates, the latter is probably the
better one because Pol is a much less processive enzyme than Pol,
suggesting that the efficiencies of the enzyme combinations differ by less
than twofold. A difference this small does not seem to provide decisive
support for the author’s hypothesis; its significance is difficult to evaluate,
both because the specific activity of the proteins is unknown and because it
176 LAWRENCE
is unclear whether the two-subunit Pol used fully reconstitutes the in vivo
activity of this enzyme. In addition to these data, Haracska and coworkers
also supported their model with a variety of genetic results, which are
discussed in Section VI.
Because the function of pol is known to be required for UV-induced
mutagenesis (reviewed in Lawrence, 2002), the role of this enzyme in
replication past UV lesions has also been investigated (Guo et al., 2001;
Johnson et al., 2000, 2001). Before the discovery of Pol, Pol was originally
described as having a modest capability for replicating past a T-T cyclobu-
tane dimer (Nelson et al., 1996a), but later work indicated a much lower
bypass frequency, and Pol is now known to be solely responsible for
replication past this lesion (Gibbs et al., 2004; Johnson et al., 1999). Such
is not the case with the T-T (6–4) UV photoproduct, which, unlike the
dimer, severely distorts local DNA structure and possesses no capability for
base-pairing. Johnson and coworkers (Johnson et al., 2001) found that
Pol was incapable of insertion opposite the 30 T of the photoproduct. It
could, however, efficiently extend from insertions carried out by either
yeast or human Pol, of which both preferentially incorporated dGMP at
this site. In the work of Guo and coworkers (Guo et al., 2001), in contrast,
Pol was found to inefficiently bypass the T-T (6–4), inserting dAMP and
dTMP, and more rarely dGMP opposite the 30 T, and inserting predomi-
nantly dAMP opposite the 50 T. Insertion at this site was most efficient
following dGMP incorporation. Pol was not examined in this study. The
apparent lack of agreement between these two investigations with respect
to Pol can probably be ascribed to differences in experimental proce-
dure; in the first, reactions contained a several-fold molar excess of
primer/template over enzymes and were terminated after 5 minutes
of incubation, whereas in the second investigation, reactions contained
a fourfold excess of enzyme over primer/template and were incubated for
30 minutes, the latter conditions favoring insertion. As discussed in section
III, in vivo experiments with yeast support the involvement of Pol in the
bypass of the T-T (6–4), but the extent of this involvement varies from
substantial to very small in different studies, the latter case raising the
question of whether Pol or some other enzyme performs insertion oppo-
site the 30 T. Such work also shows that replication past a T-T (6–4)
photoadduct in vivo is at best very inefficient, as might be expected with
such a distorting lesion, with a bypass efficiency of only 4%. As a
consequence, even enzymes that bypass this lesion only inefficiently
in vitro cannot be eliminated as candidates for this function in vivo.
In addition to UV photoproducts, a variety of DNA lesions that result
from treatment with chemical mutagens has also been examined as being
potential substrates for Pol. AAF-guanine was found by Guo et al. (2001)
to substantially inhibit this enzyme, with only a low efficiency of insertion

opposite the lesion, and very little extension beyond it, despite a fourfold
molar excess of enzyme over primer/template. However, Pol was able to
extend primers that overlapped the lesion, most efficiently with that
creating a terminal AGAAF mispair, though the correctly paired CGAAF
was extended almost as well. Pol was found to bypass both (þ) and ()
enantiomers of benzo[a]pyrene diol epoxide-deoxyguanosine adducts on
18% of templates containing these lesions. Bypass was accurate, with
insertion of only dCMP in >300 samples analyzed per lesion (Simhadri
et al., 2002). However, the bypass frequencies observed are of questionable
relevance to in vivo conditions, because the reactions lasted 90 minutes
and were carried out with high levels of the enzyme. In another study
of these lesions, and also the corresponding adducts of adenine, none of
them were detectably bypassed by Pol in reactions containing a modest
molar excess of Pol and incubation periods of up to 30 minutes
(Rechkoblit et al., 2002). The possible role of Pol and Rev1p in the bypass
of propanodeoxyguanosine, a lesion derived from acrolein that is repli-
cated accurately in vivo despite its lack of base-pairing capacity, has also
been investigated (Yang et al., 2003). Both Rev1p and Pol were found to
efficiently incorporate dCMP opposite this adduct, as needed for accurate
bypass, but were both unable to extend from this terminus, indicating that
this function might be carried out by Pol. However, Pol extended from
the inserted dCMP less efficiently than from dAMP, dGMP, or dTMP
insertions. Because Pol was unable to bypass the lesion, the basis for
the accurate replication was left unexplained. Steady-state kinetic data
suggest that Pol is also capable of extension from misinsertions of dAMP
or dTMP opposite 8-oxoG and O6-methylguanine by Pol, thought to be
involved in the bypass of these lesions because of reduced mutation
frequencies induced by MNNG in strains deleted for the Pol subunit,
Pol32p (Haracska et al., 2000, 2003). However, as discussed in Section VI, it
is doubtful that the involvement of the polymerase function of Pol can be
inferred from the pol32 mutant phenotype, and the biological relevance
of the data is questionable. Unlike its capability with other DNA lesions,
Pol can not only extend from nucleotides inserted opposite a thymine
glycol lesion but can also carry out the insertion step ( Johnson et al.,
2003). dAMP is inserted preferentially, with 13% of the efficiency for
dAMP insertion opposite undamaged thymine, and extension is also
preferentially from this incorporated residue, with 50% of the efficiency
of this process for dAMP paired with normal thymine, resulting overall in a
marked capability of the enzyme for accurate replication past this lesion.
Johnson and coworkers nevertheless suggest that Pol, rather than Pol,
performs the insertion step because it reaches the lesion first. Pol, in
178 LAWRENCE
contrast, is very inefficient at both insertion and extension with thymine

glycol. Steady-state kinetic analysis with the human and yeast Rev1p was also
used to examine insertion efficiencies opposite a variety of lesions, includ-
ing 8-oxoG, (þ)-trans-anti benzo[a]pyrene diol epoxide –N2-dG adduct,
()-trans-anti benzo[a]pyrene diol epoxide –N2-dG, acetylaminofluorene-
dG, and 1, N6-ethenoadenine with the human enzyme (Zhang et al., 2002),
and O6-methyl guanine and 8-oxoguanine with the yeast protein (Haracska
et al., 2002). In the first of these investigations, in which human Rev1p was
used, dCMP was inserted opposite a template 8-oxoG with 38% of the
efficiency of insertion opposite an undamaged template guanine, and with
15% and 18% efficiency opposite the (þ) and () benzo[a]pyrene diol
epoxide adducts, respectively. dCMP was preferentially inserted in each of
these cases. For the acetylaminofluorene-dG adduct, the efficiency relative
to template guanine was <0.1%. Interestingly, however, dCMP was inserted
opposite a template ethenoadenine adduct with an efficiency of 125%,
relative to a template adenine, suggesting that Rev1p activity may be an
important source of mutations with this lesion. In the second study, in
which yeast Rev1p was employed, insertion efficiencies opposite O6-methyl-
guanine and 8-oxoguanine lesions were found to be 2.5% and 0.25%
respectively, relative to template guanine. The marked difference between
the results for the 8-oxoG lesion in the two studies may reflect differences
between the human and yeast enzymes, but it could also result from the
influence of other factors such as sequence context.
In aggregate, these observations indicate that Pol possesses a marked
facility for extension from abnormal primer ends of many kinds. They also
show that Pol can, in some cases, insert nucleotides opposite damaged
bases, suggesting that the role of this enzyme may not be restricted
exclusively to extension. These results also confirm the strong preference
of Rev1p for dCMP insertion, even though other nucleotides can also
be used at much reduced efficiencies.
III. Genetic Analysis

Although the enzymatic properties of Pol and Rev1p in vitro and their
requirement for DNA damage induced mutagenesis in vivo strongly sug-
gest that the proteins they encode are employed in translesion replication,
this conclusion is most directly demonstrated by transformation of rev
mutant strains, with plasmid constructs containing a single defined lesion
at a specific location. Using budding yeast, such experiments yield esti-
mates of the frequency of lesion bypass and, with the aid of strains deleted
for genes encoding Pol, Pol, Rev1p, or other relevant proteins, the
relative contributions of these enzymes to replication past various lesions.
Sequence analysis of replicated plasmids identifies the nucleotide

insertions accompanying lesion bypass. This approach has been used to
investigate the roles of REV1, REV3, and REV7 in the bypass of an AAF-G
adduct, a T-T dimer, a T-T (6–4) photoadduct, and an abasic site (Baynton
et al., 1998, 1999; Bresson and Fuchs, 2002; Gibbs et al. 2004; Nelson et al.,
2000). With the exception of the T-T dimer, the bypass of each of these
lesions required the function of REV3. The function of REV7 was also
found to be necessary for the bypass of AAF-G, the only lesion examined
with this gene. However, because it is essential for Pol activity (Nelson
et al., 1996a), a requirement for REV7 is likely to follow that of REV3 in the
bypass of other types of DNA damage. These results indicate an essential
role for Pol in replication past these lesions. In the sequences examined
(50 -CACGAAFTTTC-30 , 50 -ATAGAAFCCC-30 ), bypass of the AAF-G adduct
can occur via a misaligned (slipped) primer terminus, leading to a 1
frameshift in the first sequence or þ1 frameshift in the second, or via a
correctly aligned (nonslipped) primer, resulting in accurate bypass in both
cases. Interestingly, though both of these events were abolished in rev3
or rev7 strains, only bypass via the nonslipped intermediate was substan-
tially reduced in the rev1 mutant; with the slipped intermediate, bypass
frequencies were 92% or 56% of the wild-type frequency in the two
sequence contexts examined (Baynton et al., 1999). The molecular basis
for the latter result is unclear, particularly as in other circumstances Rev1p
appears to be essential for Pol activity in vivo (Gibbs et al., 2004; Nelson
et al., 2000). In addition to requiring Pol, however, replication past the
AAF-G adduct was also found to employ Pol; in the first sequence, bypass
frequencies for both slipped and nonslipped intermediates were reduced
to a quarter of the wild-type frequency in a rad30 (Pol deficient)
mutant, and in the second sequence, they were reduced to 10% of this
frequency (Bresson and Fuchs, 2002). Pol, together with Pol and Rev1p,
was also involved in replication past a T-T (6–4) photoadduct, though the
extent of Pol involvement varied greatly in the two experiments in which
it has been investigated (Bresson and Fuchs, 2002; Gibbs et al., 2004). In
the Bresson and Fuchs study, mutagenic events bypassing the T-T (6–4)
photoadduct, which constituted 60% of the total and resulted exclusively
from the insertion of dGMP opposite the 30 T of this lesion, were reduced
to 9% of the wild-type frequency in a Pol-deficient mutant, whereas the
frequency of the remaining error-free bypass events was unaffected by
the absence of this enzyme. In the study of Gibbs and coworkers, muta-
genic bypass events with alterations at the site of the 30 T of the T-T (6–4)
lesion constituted only 15% of the total and resulted from incorpora-
tion of dCMP and dTMP as well as dGMP. In the Pol -deficient mutant,
the bypass frequency was 93% of that found in the wild type, and the
180 LAWRENCE
frequency of dGMP insertion opposite the 30 T of the T-T (6–4) lesion was
reduced from the 10% observed in the wild-type to 4% of total insertions.
In addition, mutations resulting from insertions opposite the 50 T were also
observed. Such results indicate that the intervention of Pol in the bypass
of this lesion is highly dependent on some factor such as sequence
context, which was 50 -ACAAT[6–4]TGAAC-30 in the Bresson and Fuchs
experiment, and 50 -GCAAGT[6–4]TGGAG-30 in the work of Gibbs et al.
In addition to these investigations, in which yeast strains were
transformed with plasmids that carry a specifically located lesion, similar
experiments have been carried out using transformation with lesion-
containing oligonucleotides (Otsuka et al., 2000, 2002a,b,c, 2004). This
interesting and innovative approach is based on the observation
(Moerschell et al., 1988) that yeast strains carrying a cyc1-31 mutation
can be reverted to wild-type by transformation with oligonucleotides that
have the CYC1þ sequence spanning the mutant site. The cycl-31 mutation
carries a base substitution and adjacent single-nucleotide deletion gener-
ating a stop codon, which, in the absence of transformation, reverts at only
a very low frequency. Although it is not known how the sequence present
in the single-stranded oligonucleotide is integrated into the yeast chromo-
some, it presumably requires invasion of the genomic DNA and some kind
of recombination or gene conversion event. This experimental method
therefore either achieves or closely approaches the ideal of placing a
single defined lesion at a specified genomic location. Transformation
frequencies using oligonucleotides that carried a site-specific T-T (6–4)
photoadduct were only 3% of the transformation frequencies with their
damage-free counterpart (Otsuka et al., 2002a), a value close to those
observed in the plasmid experiments; all experiments therefore indicate
that this lesion strongly blocks replication. Seventy-seven percent of the
transformants carried mutations at the lesion site, 81% of which were
30 T ! C substitutions. The frequency of these mutations was much
reduced in a rad30 strain, but the bypass frequency, though lower, was
not reduced proportionately because of a partially compensating increase
in bypass events resulting from the insertion of other nucleotides (Otsuka
et al., 2004). No transformants were found in a rev1 strain, indicating that
Rev1p is essential for the bypass of this lesion. This reflects a requirement
for the second function of Rev1p, rather than its deoxycytidyl transferase
activity, because bypass was just as efficient in a rev1 mutant strain produc-
ing protein that lacks transferase activity by virtue of D467A, E468A
substitutions (Otsuka et al., 2004). Oligonucleotide transformation was
also used to investigate the genetic requirements and mutagenic outcome
of replication past either a natural (deoxyribose) abasic site or its synthetic
tetrahydrofuran analog (Otsuka et al., 2002b). A series of isogenic strains,
comprising a wild-type and derivatives carrying deletions of APN1 or APN2

(both encoding apurinic endonucleases), RAD30, or REV1, or both APN1
and REV1, were transformed with oligonucleotides containing one or the
other of these lesions or a deoxyuracil residue. Results from oligonucleo-
tides carrying the latter base or the natural abasic site were essentially
identical, indicating that endogenous uracil-DNA-glycosylase rapidly and
efficiently removes the uracil before replication. Replication past the
natural abasic site was unchanged in the rad30 strain but was much
reduced in the rev1 mutant, indicating that Rev1p, though not Pol, is
necessary for the bypass of this lesion. In REV1þ strains, dCMP was
preferentially incorporated opposite the abasic site, with an average of
63% insertions being of this kind. This class of event was much reduced in
rev1 strains, however, showing that it results from the deoxycytidyl
transferase activity of Rev1p. Although this activity stimulates replication
past the abasic site, a reduced frequency of bypass can still occur in strains
that contain Rev1 mutant protein lacking transferase activity because of
D467A and E468A substitutions (K. Negishi, personal communication;
Otsuka et al., 2002c). Bypass of the abasic site in this strain occurs at about
half the frequency found in the wild type, a reduction that is accounted for
by the marked decrease in bypass events resulting from dCMP insertion. In
the absence of the transferase activity, the principal residue incorporated
was dTMP, though low frequencies of insertion of the other nucleotides,
including dCMP, were observed. These insertions were presumably carried
out by either Pol or Pol, both of which are no doubt less efficient at this
task than wild-type Rev1p. However, the second function of Rev1p is also
needed, as shown by the absence of bypass in the rev1 strain. This
function is probably a requirement of Rev1p for Pol activity, perhaps as
an intermediary to anchor it to PCNA (see Section V). In the presence of
wild-type Rev1, Pol, also essential for the bypass of abasic sites, can then
extend the primer to complete lesion bypass. Bypass frequencies for the
tetrahydrofuran residue were substantially lower than for the natural
abasic site in REV1þ strains, except in the apn1 strain, indicating that
the synthetic analog is a better substrate for the APN1 apurinic endonu-
clease than the natural lesion. Deletion of APN1, though not APN2, also
increased the transformation efficiencies of oligonucleotides carrying the
natural abasic site but did not alter bypass frequencies in the mutants
relative to those in the wild-type control. As with the natural abasic site,
bypass of the tetrahydrofuran residue in the apn1 strain also requires
Rev1p, as bypass is essentially abolished in the apn1 rev1 double
mutant. However, the Rev1p transferase activity appears to be much less
important with this lesion, indicating that the tetrahydrofuran abasic site is
a relatively poor substrate for dCMP insertion; 48% of the bypass events
182 LAWRENCE
result from the incorporation of dAMP, 32% from dGMP incorporation,

and only 20% from dCMP incorporation. No incorporation of dTMP was
detected. Although insertion of dCMP is much less frequent opposite the
tetrahydrofuran lesion than the natural abasic site, it is still dependent on
the Rev1p deoxycytidyl transferase activity, as such insertion is absent in an
apn1 mutant that possesses the D467A, E468A Rev1 mutant protein.
Despite this, the bypass frequency is almost unaffected by this mutant
protein (Otsuka et al., 2004). The role of Rev1p in the bypass of the
tetrahydrofuran lesion therefore appears to depend principally on its
second, Pol-supporting, function.
IV. Processes Other than General Translesion Replication

that Employ Pol and Rev1p
Although most attention has been given to the involvement of Pol
and Rev1p in translesion replication in the genome at large, these en-
zymes also appear to function in other cellular processes, most notably in
the repair of double-strand breaks and, among vertebrates, in somatic
hypermutation.
A. Somatic Hypermutation
Somatic hypermutation is the phenomenon in which a high frequency
of point mutations are generated within a 1–2-kb segment in the variable
region of expressed immunoglobulin genes in response to the presence of
an antigen. High-affinity immunoglobulins are generated by selection
among variants generated over about 20 rounds of replication, in each
of which the region specific mutation frequency is about one mutant per
kilobase, resulting in up to 2% nucleotide substitutions within the target
region overall (Diaz and Storb, 2003). These mutations are produced by
inaccurate DNA polymerases, including pol and Rev1p, that bypass damage
within the target region initiated by the activation induced cytidine deami-
nase (Muramatsu et al., 2000), which, following the conversion of cytosine to
uracil and the action of uracil-DNA-glycosylase (Di Noia and Neuberger,
2002; Rada et al., 2002), results in the production of abasic sites. Although
at least some of the GC ! AT mutations are likely to arise during
replication past uracil by accurate enzymes (Di Noia and Neuberger,
2002), the remaining substitutions presumably arise from bypass by inac-
curate bypass polymerases. Pol appears to play a major role in the latter
process. Somatic hypermutation frequencies within VHDJH and bcl-6 genes
in a human B-cell line were reduced to about 27% of control frequencies
following treatment with phosphorothioate-modified 15-mer oligonucleo-

tides that substantially reduced REV3 mRNA levels (Zan et al., 2001). A
similar result was found for 6-thioguanine resistant mutants induced in a
UV-irradiated pSP189 shuttle vector transfected into the oligonucleotide-
treated cells (Zan et al., 2001). Interestingly, Pol (REV3) transcript levels
were found to be upregulated, and Pol mRNA levels downregulated, in
cells undergoing somatic hypermutation, whereas the transcript levels for
Pol, Pol, Pol, Pol, Pol", Pol, and Pol all remained unchanged. The
involvement of Pol in somatic hypermutation is also evident in experi-
ments with transgenic mice expressing high levels of antisense RNA to
mouse REV3, which concomitantly reduced levels of endogenous REV3
mRNA transcript (Diaz et al., 2001). Mutation frequencies in memory
B-cell clones in these mice were between 56% and 72% of the frequencies
in cells from control animals, with a marked deficiency of clones that
contained more than three mutations. Such figures are likely to apprecia-
bly underestimate the role of Pol in this process. Few transgene mice were
recovered, and only 1 of 10 expressed high levels of antisense RNA,
indicating selection for animals expressing at least a minimal amount of
Pol, a conclusion also predicted from the embryonic lethality of mice
homozygous for null disruptions (Bemark et al., 2000; Esposito et al., 2000;
Wittschieben et al., 2000). As expected from its requirement for Pol
activity, Rev1p also appears to be necessary for somatic hypermutation
in DT-40 chicken cells (Simpson and Sale, 2003). Disruption of the REV1
gene in this cell line reduces nontemplated mutations, resulting from
de novo mutation, to 0.8 104 mutations per base pair, compared to a
frequency of 5.5 104 mutations per base pair in the REV1þ control.
Almost all untemplated mutations are therefore generated by a Rev1p-
dependent process, though it cannot be excluded that a small fraction
arose in a Rev1p-independent manner, as the calculated background error
frequency from the PCR procedure used was 0.4 104 mutations per
base pair. The frequency of templated changes, resulting from gene
conversion, was not decreased. Disruption of REV1 reduced the frequency
of all types of base substitutions, consistent with loss of the Pol-supporting
function of Rev1p. However, DNA sequence results from the REV1þ cell
line (Sale et al., 2001) are also consistent with the frequent employment of
the Rev1p deoxycytidyl transferase activity during somatic hypermutation.
In this clone, 51% of the mutations were C ! G or G ! C mutations, as
expected from the sequential action of the AID cytidine deaminase and
uracil-DNA-glycosylase, followed by dCMP incorporation opposite the
resulting abasic site. The remaining substitutions presumably arise from
insertions by other DNA polymerases, such as Pol, Pol, Pol, or Pol;
however, chickens do not appear to possess Pol ( J. McDonald and
184 LAWRENCE
R. Woodgate, personal communication). All of these polymerases may

be employed in somatic hypermutation to some degree in mammals. Data
from in vitro experiments suggest that Pol has a marked facility for
insertion opposite abasic sites (Vaisman et al., 2002), and a variety of
evidence indicates that Pol is employed in somatic hypermutation at
AT base pairs and certain hotspots (Rogozin et al., 2001; Zeng et al.,
2001). An extended discussion of somatic hypermutation is given in
Chapter 11 of this book.
B. Double-Strand Break Repair

Evidence for the use of Pol during the repair of double-strand breaks
has been found in budding yeast, DT-40 chicken cells, and the mouse,
though it appears to be essential for such repair only in vertebrates and is
merely associated with it in yeast. In a REV3þ diploid strain of the latter
organism, repair of a specifically located double-strand break in one of the
chromosome III homologs was accompanied by a 56- to 76-fold increase in
the spontaneous reversion rate of the trp1-488 nonsense allele located 314
base pairs from the break site. In a rev3 mutant, however, trp1-488
reversion frequency in cells repairing the double-strand break was only
1%–2% of the wild-type value, even though the induced recombination
frequency was about the same as in the REV3 strain (Holbeck and
Strathern, 1997). The reversion rates of two trp1 frameshift alleles, even
though much increased in cells repairing the double-strand break, were
not dependent on REV3, however. The types of REV3-dependent muta-
tions induced by double-strand break repair was further examined in a
subsequent investigation (Rattray et al., 2002), using forward mutations to
canavanine resistance to monitor mutagenesis rather than reversion of
trp1 alleles. As before, double-strand breaks were induced by inserting an
HO-endonuclease recognition sequence within one of the chromosome
III homologs, together with regulated expression of the endonuclease
from an HO gene controlled by the GAL1 galactose-inducible promoter.
The frequency of base-pair substitutions induced in break repair by ho-
mologous recombination was about ninefold lower in the rev3 mutant
than REV3þ strain, whereas for frame-shift mutants the decrease was only
about twofold, indicating that half of the latter events are produced by a
DNA polymerase other than Pol. Interestingly, the frequency of can1R
mutations associated with break repair by what appeared to be nonhomol-
ogous end joining rather than homologous recombination were mostly
produced by Pol, with a more than 50-fold decrease in their frequency
in the rev3 mutant compared to the REV3þ strain. This result suggests
that Pol may be employed in a greater variety of double-strand break
repair events than just homologous recombination. It would be interesting

to know whether Rev1p is also required for recombination-associated
mutagenesis in yeast.
A variety of evidence suggests that Pol, as well as participating in
translesion replication, may be directly involved in double-strand break
repair in DT-40 chicken cells, rather than just associated with it (Sonoda
et al., 2003). In these cells, a REV3/ homozygote carrying disrupted
alleles is viable but suffers a threefold higher frequency of spontaneously
occurring chromosome aberrations than its isogenic REV3þ/þ counter-
part. The REV3/ clone also exhibits higher frequencies of chromosome
aberrations induced by ionizing and UV radiations. Although, as expected
from their deficiency in translesion replication, REV3/ mutant cells are
much more sensitive than the wild-type to ionizing radiation in G1-early
S phase, they are also more sensitive in the G2 phase and, indeed, are at
least as sensitive in this phase as a DT-40 RAD54/ clone deficient in
homologous recombination (Bezzubova et al., 1997). Moreover, DT-40
REV3/ mutant cells are substantially more sensitive to ionizing radiation
than RAD18/ mutants, and a little more sensitive to UV, unlike in yeast
where rev3 mutants are much less sensitive to UV, and a little less sensitive
to ionizing radiation, than rad18 mutants (Lawrence and Christensen,
1976; McKee and Lawrence, 1979a,b).
Because Rad18p, together with Rad6p, regulates translesion replication
by Pol in yeast (Bailly et al., 1994), the greater sensitivity of REV3/
relative to RAD18/ strains in the DT-40 cells implies that Pol is em-
ployed additionally in processes other than translesion replication in
chickens. This additional process is likely to be repair of double-strand
breaks by homologous recombination because gene targeting, which de-
pends on this measure, is much reduced in the REV3/ clone, whereas
nonhomologous end joining appears to occur normally (Sonoda et al.,
2003). A DT-40 REV3/ clone has a very similar profile of sensitivities to
different DNA damaging agents as the REV3/ mutant (Simpson and
Sale, 2003), suggesting that the Rev3 and Rev1 proteins act together in this
process, as they do in translesion replication in yeast. Similarities between
the consequences of REV3/ disruptions in the mouse and DT-40 cells
suggest that mouse Pol may also be employed in double-strand break
repair. Such disruptions cause embryonic lethality (Bemark et al., 2000;
Esposito et al., 2000; Wittschieben et al., 2000), with embryonic cells
showing high frequencies of apoptosis that cannot be relieved by a disrup-
tion of p53, which also failed to rescue embryos from the lethality
(van Sloun et al., 2002). Cytological examination of cells from 11.5-day
embryos showed that disruption of REV3 led to a marked increase in
double-strand breaks and other chromosome aberrations, with 14% of
186 LAWRENCE
the cells containing these compared to 0.7% in cells from the REV3þ/þ or
REV3þ/ control clone, a result reminiscent of those from chicken DT-40
cells.
V. Regulation of Pol and Rev1p and Interactions with

Other Proteins
The regulation of Pol and Rev1p, together with the other bypass
polymerases, is poorly understood, but it presumably addresses several
problems such enzymes pose for the cell. Of these problems, a major issue
is the need to recruit the appropriate enzyme, or sequence of enzymes,
to a replication fork stalled at a particular lesion, a problem that is
particularly acute in mammals because of the number of polymerases they
potentially can employ. Another important problem is the necessity of
curtailing the activity of the enzymes in circumstances where they are not
required, to minimize the production of mutations. A variety of evidence
indicates that PCNA and Rev1p are important elements in the recruiting
problem and that specific recruiting together with tight regulation of
the amount of protein expressed and its activity is part of the means used
to limit unwanted activity in lesion-free DNA. There is, however, little
evidence in yeast for regulation of REV3 at the level of transcription, either
during the mitotic cell cycle or in response to DNA damage, where UV
irradiation led at best to only a small increase in transcript level (Singhal
et al., 1992). REV1 also appears to possess a promoter that is unresponsive
to such damage (Larimer et al., 1989). There was, however, a marked
increase in REV3 transcript in meiosis, with a maximal level that was 18-
fold above the premeiotic level, perhaps in response to a need for Pol in
the repair of the double-strand breaks responsible for homologous recom-
bination in meiotic cells. The high levels of human (Lin et al., 1999) and
mouse (Van Sloun et al., 1999) REV3 transcript in testis and ovary may
reflect the same phenomenon. Although REV3 transcript levels are only
marginally increased in UV-irradiated cells of budding yeast, more sub-
stantial responses to such treatment were observed in Neurospora (Sakai
et al., 2002, 2003). No transcripts for upr-1 (encoding Neurospora REV3),
ncrev1, or ncrev7 were detected in unirradiated cells, though they were
easily observed after 15–30 minutes in wild-type cells irradiated with 100 J/
m2 UV. An increase of mouse REV3 transcript in response to stress was also
suggested by the recovery of a partial REV3 clone from a differential screen
of a cDNA library isolated from mouse fetal cortical cells treated with
the drug pentylenetetrazol, which induces epileptic-like seizures in mice
(Kajiwara et al., 1996).
Rev3 protein levels appear to be very low in both yeast and mammals,
indicating that this may be common to all organisms. Low levels of Rev3p in
yeast appear to result, at least in part, from the presence of an out-
of-frame ATG codon, in a good context for translation (Kozak, 1986), 10
nucleotides before the open-reading frame ATG, a feature that is expected
to reduce the translation efficiency of the REV3 reading frame by a factor of
at least a hundred. Out-of-frame ATG codons in good contexts are also
present in the 50 untranslated region of the human REV3 and REV1 genes
(Gibbs et al., 1998, 2000). Moreover, 40%–50% of the human and mouse
cDNAs analyzed contained an insertion of 128 bp between nucleotides
þ139 and þ140 of the REV3 reading frame, the result of alternate splicing,
which introduces an immediate in-frame stop codon (Gibbs et al., 1998;
Murakumo et al., 2000; Van Sloun et al., 1999), reducing the proportion
of translatable transcript. These features, and perhaps others, appear to
curtail production of Rev3p and Rev1p to very low levels, presumably
to limit their inappropriate synthesis on undamaged templates.
This end is also likely to be served by mechanisms that target Pol and
other polymerases to the sites of blocked forks. As discussed at greater
length in Chapter 10, this is believed to be initiated in yeast, humans, and
perhaps all eukaryotes by modification of lysine 164 in PCNA (Hoege et al.,
2002; Stelter and Ulrich, 2003). Modification at this site acts as a molecular
mechanism to switch cells between three different modes of replication;
translesion replication, replication dependent on the error-free damage
tolerance process, and normal replication. Addition of ubiquitin to lysine
164 by the Rad6p E2 ubiquitin conjugase is thought to promote transle-
sion replication by Pol, Rev1p, Pol, and, presumably, other Y-family
enzymes. The error-free DNA damage tolerance process, on the other
hand, is promoted by polyubiquitination at lysine 164, in which ubiquitin
is conjugated to the lysine 63 residue of ubiquitin itself, using the
ubiquitin conjugase activity of the Ubc13p/Mms2p/Rad5p complex.
The addition of SUMO to lysine 164 in PCNA appears to promote
normal replication. The way these modifications of PCNA implement
the recruitment of the various DNA polymerases or other proteins is not
known, but it presumably depends, either directly or indirectly, on modi-
fication-specific association with the polymerases or some intermediate
protein. An investigation of the yeast rev6-1 mutant indicates that Rev1p
may perform such an intermediary function. The rev6-1 mutant was
isolated in a screen for strains deficient for UV-induced reversion of the
his4-38 frameshift allele (Lawrence et al., 1985c). Initial characterization
showed that rev6-1 strains were substantially deficient in the UV-induced
reversion of the ochre allele arg4-17 and the missense allele ilv1-92, as well
as of his4-38, and they were also more sensitive to UV than other rev
188 LAWRENCE
mutants. Rates of spontaneous reversion of ilv1-92 were also found to

be 10-fold higher in rev6-1 mutants than in the wild-type. Recent studies
(H. Zhang, P. E. M. Gibbs, and C. W. Lawrence, unpublished data) show
that rev6-1 mutants are incapable of replicating past a site-specific T-T
cyclobutane dimer or abasic site, and that they are also deficient for the
RAD6—dependent error-free tolerance process, but grow at normal rates.
These studies also found that the rev6-1 mutation is an allele of POL30,
encoding the sliding clamp protein PCNA, which results in a G178S
substitution in this protein, which is located in a -sheet at the interface
between monomers in this homotrimeric structure. When mapped onto
this structure, the serine side chain at this site appears to cause an
unfavorable steric interaction with tyrosine 114 in the adjacent monomer,
indicating that clamp structure may be slightly abnormal, though clearly
not sufficiently to interfere with normal replication.
Because the rev6-1 phenotype appears to indicate loss of all damage-
tolerance processes, the G178S substitution may prohibit conjugation of
ubiquitin at lysine 164, preventing recruitment of polymerases or other
proteins that promote their association with PCNA. Results of a two-hybrid
analysis (H. Zhang, P. E. M. Gibbs, and C. W. Lawrence, unpublished data)
indicate that Rev1p may be a protein of this kind. These data indicate that
Rev1p associates with wild-type PCNA, but not with the G178S mutant
protein. In addition, no association was observed between wild-type PCNA
and Rev1p containing a G193R substitution in the N-terminal BRCT
domain of this protein, a region believed to be concerned with protein–
protein interactions. The rev1-1 mutant expressing the G193R protein
retains a substantial fraction of its deoxycytidyl transferase activity but is
substantially impaired with respect to replication past a site-specific T-T
(6-4) photoadduct or abasic site (Nelson et al., 2000) and with respect
to induced mutagenesis in general (Lawrence and Christensen, 1978;
Lemontt, 1971, 1972; McKee and Lawrence, 1979a). The marked similarity
between the REV1 and REV3 mutant phenotypes, and in particular the
observation that deletion of either gene can almost interchangeably abol-
ish lesion bypass, indicate that Rev1p is required for Pol activity in vivo,
which is probably the function lost in the rev1-1 mutant. It is therefore
possible that in binding to PCNA, Rev1p recruits Pol to a replication fork
stalled at the site of a lesion. It is also possible that Rev3p alone, rather
than Rev3p/Rev7p, is bound to Rev1p in this circumstance, an association
that might enhance Pol activity. Because Rev1p is not employed in the
error-free process of DNA damage tolerance, the G178S substitution in
PCNA may also abrogate an association with proteins other than Rev1p.
In addition to regulation by means of PCNA modification, budding
yeast cells possess what appears to be a regulatory cascade initiated by a
DNA damage-sensing mechanism dependent on the checkpoint genes

RAD9, RAD17, RAD24, and MEC3. In an excision-deficient background,
deletion of any one of these genes greatly reduces UV-induced forward
mutation to canavanine resistance, down to a frequency characteristic of
rev mutants (Paulovitch et al., 1998), an observation that suggests the
existence of a signaling pathway initiated by unrepaired DNA damage that
leads to the activation or mobilization of Pol and Rev1p. Induced muta-
tion frequencies were reduced, but to a lesser extent, in an excision
proficient background.
Associations among Revp, or between them and other proteins, also
suggest the possibility of some forms of regulatory control, although the
mechanisms by which such control might be accomplished are not yet
known. Because yeast Pol activity in vitro is strongly dependent on the
binding of Rev7p to Rev3p (Nelson et al., 1996a), and human Rev7p has
been shown to associate with hRev3p (Murakumo et al., 2000), the Rev7p
subunit of Pol may perform some regulatory function. Moreover, hRev7p
associates with at least two other proteins, hRev1p and hMad2p. hRev7
binds to a site at the C terminus of hRev1p ( Murakumo, 2002; Murakumo
et al., 2001), producing a stable complex but producing no change in the
deoxycytidyl transferase activity (Masuda et al., 2003). hRev7p also binds to
the spindle assembly checkpoint protein hMad2, with which it shares
significant homology, and such an association appears to suggest regulation
tied to the cell cycle (Murakumo et al., 2001). Mouse Pol, Pol, and Pol,
each bind to mouse Revp1 at the same site as Rev7p, though, unlike the
association of Rev7p and Rev3p, their enzymatic activity appears to
be unchanged (Guo et al., 2003). Similar results have been observed with
the human counterparts of these genes (Ohashi et al., 2004). Competitive
binding to Rev1p therefore appears to play some important regulatory role
in mammals, though how this is effected is not known. As judged by
sequence comparisons, yeast Rev1p appears not to have such a binding site,
though a direct test for association between Rev1p and Pol has not yet been
reported. Last, two-hybrid screens of a human library, using different seg-
ments of hRev3p as bait, have uncovered an association of the tumor-
suppressor protein p33ING, encoded by the ING1 gene, with the amino
terminus of hRev3p (R. Murante, O. Uluckan, and C.W. Lawrence, unpub-
lished data). This protein has been shown to suppress growth and apoptosis,
promote repair of UV damage to DNA, associate with p53, and participate in
the p53 signaling pathway. Counterparts of this protein in yeast interact with
histone acetyltransferase complexes and may therefore modulate transcrip-
tion (Cheung and Li, 2001; Cheung et al., 2001; Garkavtsev et al., 1998;
Loewith et al., 2000). Although this association appears to indicate integra-
tion of Pol in a network regulating checkpoints, growth control, and other
190 LAWRENCE
aspects of responses to DNA damage, much remains to be learned about the

way this might be implemented.
VI. Conclusions and Speculations

As discussed above, there is a large amount of experimental evidence
showing that Pol and Rev1p perform important functions in replication
past a great variety of DNA lesions. There are, however, disagreements
about the particular nature of the functions of both of these enzymes.
According to one model ( Johnson et al., 2000, 2001; Prakash and Prakash,
2002), the function of Pol is exclusively to extend from insertions oppo-
site lesions, whether of the correct or incorrect nucleotide, whereas ac-
cording to another model (Lawrence, 2002; Lawrence et al., 2000), Pol
can perform both insertion and extension activities with some lesions. The
ability of this enzyme to extend from terminal mismatches ( Johnson et al.,
2000; Lawrence and Hinkle, 1996; Lawrence et al., 2000), and from
nucleotides incorporated opposite lesions by other polymerases (Guo
et al., 2001; Haracska et al., 2003; Johnson et al., 2003; Nelson et al.,
1996b), has been clearly demonstrated, but its capabilities for insertion
at lesion sites are not well established. The conclusion that Pol cannot
perform the latter function ( Johnson et al., 2000, 2001; Prakash and
Prakash, 2002) is based on in vitro assays using the two-subunit enzyme
and primed oligonucleotide templates that carry defined lesions, most of
which indicate that Pol inserts only inefficiently opposite the lesions. The
extent to which these results with the two-subunit enzyme reflect the
in vivo activity of the polymerase is still uncertain, however. It is not known,
for example, whether functional association with Rev1p, thought to occur
in cells, might alter Pol activity. In addition, incorporation opposite
lesions by Pol has been thought to be unlikely because it is believed to
be a highly accurate enzyme, having greater fidelity than Pol and nearly
the same fidelity as Pol ( Johnson et al., 2000). This conclusion was
reached by comparing error rates in the LacZ gene in M13mp2 repli-
cated by purified calf thymus Pol or Hela cell Pol (Thomas et al., 1991)
with those from a steady-state kinetic analysis of the fidelity of nucleotide
incorporation on lesion-free templates by Pol ( Johnson et al., 2000).
Because the experimental procedures were very different, the validity of
comparing these two sets of data is open to question. But, more importantly,
the data in Thomas et al. (1991) do not appear to support the conclusion
reached by Johnson and coworkers: Pol is a little less accurate than Pol,
and much less accurate than Pol. The fidelity of Pol is greater than the
fidelity of Pol by twofold or more in 7 of the 12 template base/incoming
mispaired nucleotide combinations studied, and it is approximately the
same in the remaining 5. The fidelity of Pol is at least 10-fold greater than
the fidelity of Pol overall and in the same set of comparisons, by twofold
to >700-fold; Pol does not, by these measures, appear to be an unusually
accurate polymerase, and it may in fact be the least accurate of the B-family
enzymes.
Further support for the conclusion that Pol is able to incorporate
nucleotides opposite lesions is provided by the occurrence of Pol-
independent replication past various site-specific lesions in yeast. For
example, although the proportion of Pol-independent events that bypass
a T-T (6-4) photoadduct varies in different experiments (Bresson and
Fuchs, 2002; Gibbs et al., 2004; Otsuka et al., 2002a, 2004), an appreciable
fraction of this kind is nevertheless found in each case, and in the work of
Gibbs et al. (2004) this fraction was over 90%. In this circumstance, which
polymerase might be responsible for nucleotide insertion opposite the 30 T
of the lesion? Although Pol cannot be formally excluded, the most likely
candidate is Pol. As an enzyme concerned with lesion bypass, Pol is
much better adapted than a replicase to cope with distorted templates, a
property that is demonstrated in the extension reactions it carries out.
Pol is also a better candidate because, unlike Pol, it lacks a 30 to 50
exonuclease proofreading function, which could inhibit nucleotide inser-
tion. Such an effect is seen with E. coli DNA polymerase III. In cells
lacking all other DNA polymerases, this enzyme can bypass a T-T cyclobu-
tane dimer, but only if proofreading is disabled by the mutD5 mutation
(Vanderwiele et al., 1998). Pol is also a much more suitable enzyme for
incorporation because, as discussed above, it is a much less accurate than
Pol, again consistent with a relaxed requirement for normal template
structure. For the same reasons, Pol is a good candidate for insertion
opposite the 30 T of a T-T cyclobutane dimer in Pol-deficient cells, which
results in an error rate of 9%, an error rate atypical of a replicase (Gibbs
et al., 2004). Pol is also likely to be responsible for the bypass of abasic
sites that is Pol and Rev1p independent (Gibbs et al., 2004).
A second major difference of interpretation concerns the question of
whether the Rev1p deoxycytidyl transferase activity plays any part in the
bypass of an abasic site in yeast. Although investigations using plasmids
and oligonucleotides carrying a specifically located natural abasic site all
show that bypass of this lesion in yeast is chiefly accomplished by Rev1p-
dependent insertion of dCMP (Gibbs et al., 2004; Nelson et al., 2000;
Otsuka et al., 2002b) Haracska and colleagues (2001c) have concluded
that such bypass depends principally on the incorporation of dAMP by
Pol, followed by extension from this terminus by Pol. In addition to the
relative efficiencies of the Pol/Pol and Pol/Rev1p combinations for
bypass of an abasic site in vitro (see section IIB), evidence cited by these
192 LAWRENCE
authors to support this model includes results from MMS-induced muta-

genesis in an apn1 apn2 mutant, a critique of plasmid-based methods,
and inference from the phenotype of the pol32 mutant. The interpreta-
tion of each of these pieces of evidence is open to question, however, and
the case made does not seem compelling.
Evidence interpreted as showing preferential insertion of dAMP, and
not dCMP, opposite abasic sites in the genome was obtained by treating an
apn1 apn2 yeast strain with MMS, followed by sequence analysis of the
forward mutations to canavanine resistance (canR), which this treatment
induced. Based on the spectrum of base-pair substitutions induced, and
assuming that these resulted from abasic residues at sites of template
guanine or adenine, it was calculated that 64% of the mutations resulted
from dAMP insertion, with lesser and approximately equal frequencies of
insertions of the remaining nucleotides. A basic problem with this proce-
dure is that it is incapable of providing unbiased estimates of nucleotide
insertion frequencies because it only detects mutagenic events and cannot,
unlike experiments with site-specific abasic sites, estimate the frequencies
of insertions that restore the correct sequence. In particular, it cannot
detect the insertion of dCMP opposite the site of a lesion derived from a
template guanine, the predominant event expected with Rev1p.
This is likely to have resulted in a serious underestimation of dCMP
insertion in the experiment of Haracska and coworkers (2001c) because
79% (22/28) of the base substitutions sequenced resulted from alterations
at GC sites, implying that the majority of abasic sites arose from the loss
of a guanine base. At the same time, it is unclear how many of the
mutations were in fact induced by abasic residues, rather than by methyla-
tion adducts or other kinds of DNA damage. Abasic sites are likely to be
highly labile in the absence of the protection provided by bound AP
endonucleases (Mol et al., 2000), and they probably undergo a spontane-
ous -elimination reaction, resulting in a nicked strand that is a substrate
for repair rather than replication, providing a greater opportunity for
mutagenesis by alkylated bases that have escaped repair.
As a further argument against preferential incorporation of dCMP
opposite abasic sites in yeast, Haracska and coworkers cite results from
another plasmid-based experiment (Kunz et al., 1994) that appeared to
indicate preferential insertion of dGMP. A reevaluation of these data in
the light of later work indicates, however, that the results may not in fact
be inconsistent with dCMP insertion, again because procedures that mon-
itor insertion events by selecting mutations, as used in this experiment, fail
to detect events that restore the wild-type sequence. At the same time, the
identity of mutagenic lesions is not well defined in these experiments,
unlike those using the site-specific techniques. In the experiment of Kunz
and coworkers, the basis for the mutator phenotype in an apn1 deletion
mutant of yeast was investigated by sequence analysis of spontaneous
mutations that inactivate the SUP4-o tRNA suppressor, carried on a plas-
mid. Because spontaneous mutation was much reduced in an apn1
mag1 strain, such mutations were thought to have arisen at abasic sites
produced by the action of N3-methyladenine (Mag1) glycosylase on en-
dogenously alkylated adenine. Because the largest increase of spontaneous
mutations in the apn1 mutant were AT ! CG substitutions, most of the
mutations were consequently interpreted as having arisen from the inser-
tion of dGMP, rather than dCMP opposite abasic sites generated in this
way. However, the work of Guillet and Boiteux (2003) indicates that a high
proportion of the abasic sites may have arisen not by removal of adenine
but by removal of deoxyuracil that was inserted opposite template adenine
in the apn1 strain, indicating that dCMP, rather than dGMP, incorpora-
tion was indeed the major event, exactly as found with the site-specific
abasic residue. Guillet and Boiteux observed that the lethality of an apn1
apn2 rad1 yeast strain can be relieved by an ung1 mutation, deficient
in uracil-DNA-glycosylase, or by overexpression of DUT1, encoding the
dUTP pyrophosphatase, but not by a deletion of MAG1, OGG1, NTG1, or
NTG2. This result indicates that many of the highly toxic abasic sites in an
apn1 strain arise from incorporation of dUTP opposite a template
adenine in the genome, followed by its removal by uracil-DNA-glycosylase.
Moreover, abasic sites may have also arisen at abasic sites generated by
removal of template guanine. In these cases, insertion of dCMP cannot be
detected by the SUP4-o system, because it restores the normal base-pair
rather than generating a mutation. Removal of guanine is predicted
because N3 methyladenine glycosylases, of the kind encoded by yeast
MAG1, discriminate relatively poorly between alkylated and normal bases,
principally releasing guanine from nonalkylated DNA, though also releas-
ing adenine and pyrimidines at fivefold and 20-fold lower frequencies,
respectively (Berdal et al., 1998). As a consequence, overproduction of
Mag1 leads to a strong mutator phenotype (Glassner et al., 1998), and
some guanine release is likely even when Mag1 expression is normal. It is
far from clear, therefore, that the results of Kunz et al. (1994) are in fact
different from those using plasmids carrying a site-specific abasic residue.
Lastly, Haracska and coworkers implicate Pol in the bypass of abasic
sites because deletion of POL32, which encodes a nonessential subunit
of this enzyme, essentially abolishes the induction of canR mutations by
MMS. This observation is unlikely to indicate the involvement of the
catalytic function of Pol in the bypass of abasic sites, however, because
such a function is by necessity present in the pol32 strain. More probably,
the loss of the Pol32 subunit has an indirect influence on translesion
194 LAWRENCE
replication, dependent on its association with the Srs2 helicase, as de-

tected by two-hybrid analysis (Huang et al., 2000). Srs2 disrupts the Rad51
nucleoprotein filament (Krejci et al., 2003; Veaute et al., 2003), thereby
preventing homologous recombination repair and presumably maintain-
ing the blocked fork structures as substrates for RAD6-dependent DNA
damage tolerance activities. In the absence of Srs2, blocked forks are
diverted into the Rad52-dependent recombination repair pathway
(Schiestl et al., 1990), preventing processing by the Rad6 pathway, and
hence the generation of mutations. It is probably for this reason that
the frequency of induced mutations is reduced in both srs2 and pol32
mutants (Aboussekhra et al., 1989, 1992).
Although incorporation of dCMP by Rev1p appears to be the major
event during bypass of an abasic site in yeast, such a preference is less
common in vertebrates, perhaps because a greater variety of DNA poly-
merases may participate in this event. Of these, Pol in particular has a
marked facility for insertion opposite this lesion (Vaisman et al., 2002),
and the efficiency of this enzyme may out-compete that of Rev1p. In some
cases (Avkin et al., 2002; Takeshita and Eisenberg, 1994), the absence of a
dCMP preference may well result from the use of synthetic analogs of
natural abasic sites; as shown by Otsuka and coworkers (2002b), such
analogs are poor substrates for the deoxycytidyl transferase activity of
Rev1p. Nevertheless, preferential incorporation of dCMP is not necessarily
observed in mammalian cells even when a natural abasic site is used
(Cabral-Neto et al., 1994). The maintenance throughout evolution of the
Rev1p transferase activity in mammalian cells, even though it does not
appear to be employed, is puzzling: perhaps altered levels of expression of
the various Y-family enzymes, uncharacteristic of the levels in the animal,
occurred during establishment of the cell lines, an issue for further
investigation. However, preferential incorporation of dCMP opposite aba-
sic sites is found in at least one vertebrate system; namely, the immuno-
globulin genes of DT-40 chicken cells (Sale et al., 2001; see Section IV.A),
in which natural abasic sites are generated by the sequential action of the
activation-induced cytidine deaminase and uracil-DNA-glycosylase. Al-
though it is perplexing that mammalian cells can maintain a deoxcytidyl
transferase activity, but not apparently use it, it is also puzzling why
eukaryotes should possess such an activity in the first place. Why do
enzymes in the Rev1 branch of the Y family only use dCTP, and not all
four dNTPs, as do members of the other branches of this family, from
which the Rev1p branch was presumably evolutionarily derived? One
possible explanation is that restricting incorporation to dCMP, although
inevitably causing mutations at abasic sites previously occupied by nucleo-
tides other than guanine, nevertheless still results in a lower mutation
frequency than would occur from insertion by DNA polymerases that use
all four dNTPs. It will be interesting to identify the particular residues and
structural features of Rev1p that exclude incorporation of dNTPs other
than dCTP, structures that are presumably absent in the members of the
other branches of the Y family.
Finally, perhaps the greatest problem with enzymatic studies of Pol and
Rev1p concerns the properties and structure of the native enzymes. As in
the case of Pol, for which a considerable enhancement of insertion
efficiency was observed following association with PCNA and the presence
of RPA (Haracska et al., 2001b,d), the properties of Pol and Rev1p may
also be enhanced by such factors. In particular, the apparent inefficiency
of Pol for insertion opposite lesions in vitro may reflect their absence. A
variety of evidence suggests that Pol and Rev1p are associated with one
another in a multiprotein complex of some kind, possibly because Rev1p
acts as a structural link between Pol and PCNA (see Section V). Identify-
ing and investigating the enzymatic properties of such a complex is likely
to be needed for a fuller characterization of these proteins.
Acknowledgments
This work was supported by Public Health Service grant GM60652 from the National
Institutes of Health.
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(1998). Identification, chromosomal mapping and tissue-specific expression of
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(2003). Mammalian translesion DNA synthesis across an acrolein-derived deoxy-
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DNA POLYMERASES h AND i
By ALEXANDRA VAISMAN,* ALAN R. LEHMANN,À AND ROGER WOODGATE*
*Laboratory of Genomic Integrity, National Institute of Child Health and Human Development,
National Institutes of Health, Bethesda, Maryland, 20892-2725
À
Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ,
United Kingdom
I. Historical Perspective . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 205

II. Identification of RAD30 and its Orthologs. . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 206
III. Biochemical Properties of Pol and Pol . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 207
IV. Translesion Synthesis by Pol and Pol . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 210
V. Structure of the Catalytic Core of S. Cerevisiae Pol .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 212
VI. Regulation and Localization of Pol and Pol. . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 215
VII. Mutations in Pol in XP Variants. . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 217
VIII. Pols and and the Polymerase Switch: Interactions with PCNA and Rev1 . . . 219
IX. Protection from Cellular Effects of DNA Damage . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 220
X. Roles of Pol and Pol in Somatic Hypermutation . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 221
References. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 222
I. HISTORICAL PERSPECTIVE
Based on genetic studies in Escherichia coli, it was believed for many years
that damage-induced mutations in DNA were generated in a two-step
process commonly referred to as Translesion DNA Synthesis (TLS) (for
a general review, see Friedberg et al., 1995). The first step of this process is
(mis)incorporation and involves the physical insertion of a nucleotide
opposite the DNA lesion. The second step involves extension of the
(mis)incorporated base, such that the replication-blocking lesion is
completely bypassed. It was originally hypothesized that both steps were
performed by the cell’s high-fidelity replicase, Pol III, with the assistance of
accessory proteins, such as RecA and UmuDC, which together facilitated
highly error-prone DNA replication through various DNA lesions (Bridges
and Woodgate, 1984, 1985a,b).
The first clue that cells may actually contain specific polymerases
specialized in lesion bypass came in 1996 with the purification of Pol, a
heterodimer consisting of Rev3/Rev7, which has the ability to replicate past
ultraviolet (UV) induced thymine–thymine cis–syn cyclobutane dimers
(CPDs) (Nelson et al., 1996b) (reviewed in detail in Chapter 6). At the
same time, it was found that the product of the Saccharomyces cerevisiae REV1
gene long implicated in the mutagenic process has deoxycytidyl transferase
activity (Nelson et al., 1996a). Although the REV3 gene encoding the
catalytic subdomain of Pol belongs to the B-family of DNA polymerases,

206 VAISMAN ET AL.
the REV1 gene exhibits no sequence or motif homology to any of the

previously identified DNA polymerase families. Instead, it shares homology
with the umuC gene (Larimer et al., 1989) that is required for damage-
induced mutagenesis in E. coli (Kato and Shinoura, 1977; Steinborn, 1978).
Because at that time Rev1 was believed to use only dCTP as a substrate, the
possibility that UmuC or phylogenetically related proteins might actually
possess DNA polymerase activity was largely overlooked. However, that view
began to change in the late 1990s, when it was shown that a highly purified
preparation of heterodimeric E. coli UmuD0 2C was able to bypass a synthetic
abasic site unassisted (Tang et al., 1998). Although at that time there was a
possibility that the UmuD0 2C preparation contained a trace amount of
endogenous PolIII, the biochemical studies on UmuD0 2C provided the first
hint that many of the proteins implicated in the mutagenic process might
actually be bona fide DNA polymerases. The intrinsic polymerase activity of
the UmuD0 2C complex was subsequently confirmed (Tang et al., 1999) and
was shown to reside in the UmuC subunit of the complex (Reuven et al.,
1999). Amazingly, within a short, 18-month period, DNA polymerase
activity was also described for several proteins homologous to UmuC and
Rev1. These include E. coli PolIV, encoded by the dinB gene (Wagner et al.,
1999) and described in detail in Chapter 8; its human homolog Pol
( Johnson et al., 2000a; Ohashi et al., 2000; Zhang et al., 2000c), described
in Chapter 9; and two eukaryotic paralogs of S. cerevisiae RAD30, Pol
( Johnson et al., 1999b; Masutani et al., 1999) and Pol ( Johnson et al.,
2000b; Tissier et al., 2000b; Zhang et al., 2000b), which are described in
detail later. Collectively, these proteins are now referred to as the Y-family of
DNA polymerases (Ohmori et al., 2001).
Over 200 proteins that share homology to the Y-family polymerases have
since been identified in prokaryotes, archaea, and eukayrotes. Interesting-
ly, many organisms often possess more than one family member, suggest-
ing that Y-family polymerases play important roles in cellular survival or
evolutionary ‘‘fitness’’ (Friedberg et al., 2002; Yeiser et al., 2002). Indeed,
defects in human Pol result in the sunlight-sensitive and cancer-prone
Xeroderma pigmentosum variant (XP-V) syndrome ( Johnson et al., 1999a;
Masutani et al., 1999) (see following), whereas mutations in E. coli dinB
reduces the cell’s ability to undergo adaptive mutagenesis in stationary
phase (McKenzie et al., 2001; Tompkins et al., 2003).
II. IDENTIFICATION OF RAD30 AND ITS ORTHOLOGS

The RAD30 gene was identified in a search of the S. cerevisiae genome for
homologs of prokaryotic DinB- and UmuC-like proteins and for eukaryotic
Revl-like proteins (McDonald et al., 1997; Roush et al., 1998). Similar to its
DNA POLYMERASES AND 207
E. coli counterparts, RAD30 is damage inducible, and Rad30-disrupted

yeast strains are mildly sensitive to UV light (McDonald et al., 1997; Roush
et al., 1998). Epistasis analysis suggested an involvement of the Rad30
protein in the postreplication repair pathway (McDonald et al., 1997),
and an important step in understanding the cellular function of Rad30
came with its overproduction and purification ( Johnson et al., 1999b).
Biochemical characterization revealed that the Rad30 protein is a bona fide
DNA polymerase, which uses all four nucleoside triphosphates in a tem-
plate-dependent reaction. Strikingly, however, Rad30 was shown to bypass
a thymine–thymine CPD exceptionally efficiently and relatively accurately
( Johnson et al., 1999b). As the sixth eukaryotic polymerase reported in the
literature at that time, the S. cerevisiae Rad30 protein was designated
as Pol.
Shortly afterward, a human homolog of RAD30 was identified and
shown to be the enzyme mutated or missing in cells from humans exhibit-
ing the XP-V phenotype ( Johnson et al., 1999a; Masutani et al., 1999). XP-V
cells are proficient in nucleotide excision repair but defective in postre-
plicative repair (they have a reduced ability to make intact daughter DNA
strands on damaged templates) (Lehmann et al., 1975). This defect results
in an increase in UV-induced mutagenesis and leads to a high incidence
of skin cancer (Cleaver and Carter, 1973). The name approved by the
Human Genome Organization for the human RAD30 ortholog is POLH,
although it is also commonly referred to as the XPV or RAD30A gene.
Soon after the discovery of human POLH, a second human homolog of
RAD30 was identified and initially designated RAD30B (McDonald et al.,
1999). Subsequent studies demonstrated that, similar to the other phylo-
genetically related proteins, the Rad30B protein has DNA polymerase
activity. The protein was therefore renamed DNA polymerase and is
encoded by the human POLI gene (Tissier et al., 2000b).
POLH orthologs are evolutionally conserved in a wide variety of eukar-
yotes from yeast to human, and it is thought that POLI most likely arose as
a gene duplication of POLH during evolution of the species. POLI ’s
distribution is much more limited than that of POLH, and it is found
mostly in higher eukaryotes.
III. BIOCHEMICAL PROPERTIES OF POL AND POL

Similar to other members of the Y-family, both Pol and Pol are highly
error prone and lack intrinsic exonuclease activity, meaning they cannot
proofread any of the errors they make when copying DNA. Both enzymes
are distributive and incorporate only a few deoxynucleotides before
dissociating from the extended product. There are, however, significant
208 VAISMAN ET AL.
differences in the biochemical properties of these phylogenetically related

enzymes. For example, a property that distinguishes human Pol from Pol
and other members of Y-family DNA polymerases is its intrinsic 50 -deoxyri-
bose phosphate (dRP) lyase activity (Bebenek et al., 2001b; Prasad et al.,
2003); that is, the ability to catalyze excision of 50 -dRP from DNA during
base-excision repair. Furthermore, Pol has stronger dRP lyase activity
relative to its polymerase activity than does the main BER polymerase,
Pol (Prasad et al., 2003).
The fidelity of both yeast and human Pol is very low, with in vitro
misincorporation frequencies ranging from 102 to 103 (Fig. 1A; Johnson
et al., 2000c; Matsuda et al., 2000; Washington et al., 1999), which is at least
100 times less accurate than that of replicative DNA polymerases (Kunkel,
2004). As is usually seen with other DNA polymerases, error rates vary
depending on mismatch composition and template sequence context
(Goodman and Fygenson, 1998; Goodman et al., 1993; Mendelman et al.,
1989; Zhang and Mathews, 1995). For example, higher misincorporation
rates are observed when the base-pair 50 of the error is T:A or A:T
compared to C:G or G:C (Matsuda et al., 2000). Pol’s misincorporation
frequency and specificity is unprecedented, even when compared to Pol.
In general, Pol forms most mispairs more frequently than Pol (Fig. 1A).
Furthermore, Pol is characterized by a significantly broader range of
misincorporation frequencies for different mispairs compared to Pol
(Fig. 1, compare distribution of points along x - and y -axes).
The fidelity of Pol is uniquely template dependent. On primed single-
stranded DNA, the most accurate nucleotide incorporation is observed
opposite template A (with a misinsertion frequency of about 1 104)
(Tissier et al., 2000b). The preference for the correct nucleotide in this
case is ensured by tighter binding and faster incorporation compared to
the binding and incorporation of the incorrect nucleotide (Washington
et al., 2004). In contrast, Pol makes an extremely high level of errors at
template T. In fact, depending on the sequence context, the wobble base,
G, is incorporated 3 to 11 times more often opposite T than the Watson–
Crick base, A ( Johnson et al., 2000b; Tissier et al., 2000b; Vaisman et al.,
2001). On DNA templates with a 1-nucleotide 50 overhang, the pattern of
nucleotide misincorporation by Pol is entirely different from that on
primed single-stranded DNA templates. In this case, Pol is most inaccu-
rate on template C, where C and A are misinserted three- to eight-fold
more often than the correct base, G (Frank et al., 2001).
Pol and Pol are characterized not only by high levels of nucleotide
misinsertion but also by their relatively high efficiency of mismatch exten-
sion compared to classical DNA polymerases. The range of single mismatch
extension frequencies by Pol and both yeast and human Pol are similar
FIG. 1. Comparison of insertion and extension fidelities of human Pols and .

(A) Efficiencies of misincorporation ( finc) by Pol (Tissier et al., 2000b) were plotted
versus efficiencies of misincorporation by Pol ( Johnson et al., 2000c; Washington et al.,
1999), which is at least 100 times less accurate than that of replicative DNA polymerases
(Kunkel, 2004 #1346; Washington et al., 1999). The dashed line corresponds to
finc Pol ¼ finc Pol . Points above the dashed line correspond to a higher frequency of
nucleotide misinsertion catalyzed by Pol compared with that of Pol, whereas points
below the dashed line represent the reverse situation. In the base mispairs shown, the
first base is the incoming nucleotide and the second base is in the template. (B)
0
Efficiencies of mispair extension ( fext ) by Pol (Vaisman et al., 2001) were plotted versus
the efficiencies of mispair extension by Pol (Washington et al., 2001). The dashed line
corresponds to f 0ext Pol ¼ f 0ext Pol . Points above the dashed line correspond to a higher
relative efficiency of mispair extension catalyzed by Pol compared with that of Pol,
whereas points below the dashed line represent the reverse situation. In the base
mispairs shown, the first base at the 30 primer terminus and the second base is in the
template.
(Fig. 1B; Vaisman et al., 2001; Washington et al., 2001). Mismatch extension
by human Pol is somewhat less efficient than the incorporation of the
wrong nucleotide for most mispairs (Fig. 1; Washington et al., 2001). For
Pol, the correlation between relative efficiencies of mismatch formation
and extension is more dependent on the identity of a mispair and the next
template nucleotide encountered (Fig. 1; Vaisman et al., 2001). More mis-
pairs are extended by Pol, with a higher efficiency than by Pol (Fig. 1B;
Matsuda et al., 2000; Vaisman et al., 2001; Washington et al., 2001). Although
Pol is able to extend a variety of mismatches quite efficiently, a ‘‘buried’’
mispair located 2–3 bases from the nascent primer chain significantly
inhibits further primer elongation and therefore restricts Pol synthesis
to short regions of DNA (Vaisman et al., 2001). Pol is also essentially
210 VAISMAN ET AL.
blocked by tandem mispairs, whereas human Pol extends tandem double-

mismatched termini at rates similar to those at which other polymerases
extend single mismatches (Matsuda et al., 2000). Human Pol also generates
a remarkable variety of nucleotide deletion and addition errors with high
frequencies (Matsuda et al., 2000). These frameshift errors are explained by
strand slippage, often preceded by nucleotide misincorporation.
IV. TRANSLESION SYNTHESIS BY POL AND POL

The ability of Pol and Pol to bypass a variety of structurally diverse
and potentially mutagenic lesions in vitro has been analyzed extensively.
These biochemical studies revealed that the efficiency and pattern
of nucleotide incorporation opposite DNA damaged sites is distinct for
the two Rad30 paralogs. In some cases, Pol readily bypasses a lesion,
whereas Pol does it inefficiently, or not at all. An example of Pol’s
and Pol’s disparate TLS activities is observed at thymine CPDs and
cisplatin-induced dG intrastrand crosslinks. Pol efficiently facilitates ro-
bust bypass of both CPDs ( Johnson et al., 2000c; Masutani et al., 2000;
McCulloch et al., 2004) and platinum-GG cross links (Vaisman et al., 2000)
in vitro, whereas Pol bypasses the CPD in a limited manner (Tissier et al.,
2000a) that is sequence context dependent (Vaisman et al., 2003) and
is incapable of nucleotide incorporation opposite a cisplatin adduct
(McDonald et al., 2001).
On the contrary, Pol appears to be limited to insertion opposite the
30 T of UV-induced 6-4 pyrimidine-pyrimidone dimers (6-4PP) and is
significantly inhibited after nucleotide incorporation at abasic sites and
N-2-acetylaminofluorene (AAF)-modified guanine adducts (Haracska et al.,
2001c; Kusumoto et al., 2002; Zhang et al., 2000a), whereas Pol is signifi-
cantly more efficient at incorporating across from both Ts of the 6-4PP
(Tissier et al., 2000a; Vaisman et al., 2003) and opposite an abasic site
( Johnson et al., 2000b; McDonald et al., 2001). When encountering certain
other lesions, the enzymes behave in a similar manner. For example,
both Pols can readily bypass an 8-oxoG lesion (Vaisman and Woodgate,
2001; Zhang et al., 2000a), and both are limited to inefficient incor-
poration opposite acrolein-derived deoxyguanosine (Minko et al., 2003;
Yang et al., 2003) and benzo[a]pyrene (BP) adducts (Chiapperino et al.,
2002; Frank et al., 2002; Rechkoblit et al., 2002). Thus, TLS not only
depends on the particular lesion encountered but also on the polymerase
attempting TLS.
The accuracy with which any given lesion is bypassed is also uniquely
dependent on each polymerase. The fact that humans lacking Pol have
an increased incidence of skin cancers clearly indicates that Pol plays an
important role in the accurate postreplication repair of UV-induced

CPDs in vivo. Depending on the point of view taken, the fidelity of bypass
of CPDs in vitro by Pol can be regarded as error prone or relatively
error free. Using a template containing a CPD, ‘‘incorrect bases’’ opposite
the Ts are inserted at a frequency of approximately 102 ( Johnson et al.,
2000c; Masutani et al., 2000; McCulloch et al., 2004), which could be
regarded as error prone. However, this means that in 99% of inst-
ances, Pol inserts the ‘‘correct’’ bases opposite these lesions. No other
polymerase displays any comparable accuracy.
With other lesions, Pol frequently misinserts erroneous bases. For
example, dG is predominantly misinserted at the 30 T of the 6-4PP before
DNA synthesis is aborted ( Johnson et al., 2001; Masutani et al., 2000). Pol
also predominantly misincorporates dA, and to a lesser degree dG and dT,
at acrolein-derived dG (Yang et al., 2003), dA at cyclodeoxyadenosine
(Kuraoka et al., 2001), dT at O6-methylguanine (Haracska et al., 2002),
and dG and dA opposite BP-G adducts (Chiapperino et al., 2002). On the
basis of highly error-prone nature of the enzyme at these specific lesions
in vitro, one has to speculate that either these errors are removed in vivo by
exonucleolytic proofreading (Bebenek et al., 2001a) or TLS of the lesions
is facilitated by a more accurate enzyme.
The specificity of nucleotide misincorporation opposite many of the
lesions tested is very different for Pol compared to that of Pol. Pol is
characterized by unique nucleotide selectivity for most of the damaged
sites and often incorporates the wrong nucleotide more efficiently than
the correct one. Despite the fact that coding properties of damaged bases
essentially depend on chemical structure, the miscoding potential of DNA
lesions for Pol appears to be related to its fidelity on undamaged DNA.
The selection of nucleotides by Pol is most unpredictable at template T.
Depending on the surrounding sequence context and the type of damage,
A, T, or G is predominantly inserted. An unusually accurate incorporation
is observed at the 30 T of 6-4PP in an ATTC sequence context (Vaisman
et al., 2003). This exception is particularly interesting because structural
features of 6-4PP-containing duplex DNA suggest that 30 T of 6-4PP forms a
more stable base pair with G than with A (Kim and Choi, 1995; Lee et al.,
1999), which is consistent with the misincorporation specificity of other
DNA polymerases, including Pol ( Johnson et al., 2001).
In contrast to the extremely promiscuous behavior of Pol on template
T, the enzyme is very accurate at template A, which is either undamaged or
contains various stereoisomers of BP adducts placed in different sequence
contexts (Frank et al., 2002). Although the misincorporation frequency
increases upon formation of BP adducts at adenosine, the fidelity of Pol
remains high (Frank et al., 2002). Thus, it is possible that while Pol has
212 VAISMAN ET AL.
evolved to protect us from damage occurring at thymine, Pol may simi-

larly help protect us from mutations at damaged adenosines.
In addition to yeast and human Pol and human Pol, Drosophila and
mouse Pol and Pol enzymes have been overproduced and characterized
(Ishikawa et al., 2001; Matsuda et al., 2001; McDonald et al., 2003).
Although the enzymatic properties of the various Pols were similar from
all sources, the properties of Drosophila Pol were very different from either
the human or mouse Pol enzymes. In fact, Drosophila Pol is more akin to
Pol in its ability to traverse a CPD accurately (Ishikawa et al., 2001). Such
observations lend support to the notion that Pol arose as a duplication of
Pol and that evolutionary selective pressures acted on Pol over the
millennia, so as to distinguish it from Pol.
V. STRUCTURE OF THE CATALYTIC CORE OF S. CEREVISIAE POL

Although human Pols and are only 23% identical to each other at
the primary sequence level, they nevertheless have conserved blocks of
amino acids (called motif I–V in Kulaeva et al., 1996), which are readily
identifiable when their primary amino acid sequences are aligned to other
Y-family polymerases (Fig. 2A). These five motifs are located in the
N-terminal half of the two proteins, which is now known to contain the
catalytic active site of all Y-family polymerases (Ling et al., 2001; Silvian et al.,
2001; Trincao et al., 2001; Zhou et al., 2001). Remarkably, even though
there is little to no similarity in the primary amino acid sequence of Y-
family polymerases with those previously identified from the A-, B-, C-, D-,
or X-polymerase families, the three-dimensional structure of Pol is similar
to those previously reported for other DNA polymerases, in that it retains
the overall topology of a right hand that is capable of holding DNA in its
grasp (Fig 2B). Similar to the classical polymerases, the catalytic core of
Pol is composed of a thumb subdomain, which is thought to have a
primary role in DNA duplex binding and polymerase processivity; a fingers
subdomain, which is likely to be important for nucleoside triphosphate
(dNTP) selection; and a palm subdomain, which contains the catalytically
important residues for the nucleotidyl transfer reaction.
The core of the palm domain of Pol includes a four-stranded antipar-
allel sheet flanked by two small helices and an -helical structure
situated at the base of the palm, which is superimposable with the core of
the palm domain from high-fidelity polymerases. Akin to the active sites
of the polymerases from the A-, B-, C-, and X-families, the palm domain of
Pol contains three carboxylates (Asp30 and Asp155-Glu156), which coor-
dinate two catalytically essential metal ions assisting the nucleotidyl trans-
ferase reaction. Although the palm subdomain is nearly superimposable
FIG. 2. Cartoon representation of the conserved regions in Pols and and the
crystal structure of the catalytic core of Saccharomyces cerevisiae Pol. (A): Schematic
alignment of hPOLH and hPOLI. Conserved amino acid sequences found in all Y-family
polymerases are represented by the five colored boxes (motifs I–V) and by white boxes
indicating unique sequences. Gaps have been introduced in the sequences for optimal
alignment. The length of Pols and are indicated by the number of amino acids on
the right site of the diagram. A conserved zinc-binding motif in the C terminus of Pol
is indicated as C2H2 in the gray box. Structural domains of Pol shown in blue (finger,
F), red (palm, P), green (thumb, T), and purple (little finger, LF) are indicated above
the alignment. The positions of the nuclear localization signal (NLS), the domain
involved in polymerase localizing into replication foci (FS), and regions responsible for
the interaction with other proteins are also indicated. (B) Structural domains of Pol
are shown in red (palm), green (thumb), blue (finger), and purple (little finger) as a
ribbon diagram. The acidic residues (Asp30, Asp155, and Glu156) that make up the
active site are shown as gold rods. S. cerevisiae Pol has an insert of 70 amino acids in
the palm domain that is absent in the archaeal Y-family polymerases (Ling et al., 2001;
Silvian et al., 2001; Zhou et al., 2001), and this region is shown in pink. This figure was
made with ribbons (Carson, 1987). (See Color Insert.)
214 VAISMAN ET AL.
on the palm of high-fidelity polymerases, the fingers and the thumb

domains of Pol are small and stubby when compared to the fingers
and thumb of polymerases from other families. These structural features
provide a basis for the tolerance by Pol for mismatch- or lesion-induced
geometric distortions in the DNA. The fingers subdomain in Pol consists
of a -sheet and three small helices (Fig. 2). The ‘‘O helices,’’ which are
believed to play a key role in fidelity enhancement by tightening the
interface between the polymerase and the replicating base pair, are absent
in the fingers of Pol. Instead, only a short loop is positioned to potentially
interact with the replicating base pair. The thumb subdomain of Pol is
composed of six helices that are structurally distinct from helices in
other DNA polymerases (Fig. 2B).
The surface area in the Pol active site enclosed by the palm, fingers,
and thumb domains is reduced compared to classical polymerases
and appears to be too small to allow for efficient binding and replicating
DNA. To increase its binding area, Pol employs an additional C-terminal
domain, which mimics an extra set of fingers and has been termed
the polymerase-associated domain (PAD) (Trincao et al., 2001). This
domain is not found in DNA polymerases from other families but
is structurally conserved within the Y-family polymerases. In archaeal DinB
homologs Dpo4 and Dbh, it has been termed the ‘‘little finger’’ (Ling et al.,
2001) and the ‘‘wrist,’’ respectively (Silvian et al., 2001), and we will refer
to this domain as the little finger (LF) throughout the rest of this chapter.
The LF domain has been proposed to play a role similar to that of the
accessory factors employed by other polymerases, so as to increase the
processivity of the polymerase (Boudsocq et al., 2004). This domain in Pol
consists of an antiparallel sheet and two long helices packed next to
the fingers subdomain and is connected to the thumb by an extended
peptide (Fig. 2B).
The crystal structure of S. cerevisiae Pol lacked 120 C-terminal residues,
which, although unnecessary for polymerase activity in vitro, are likely to be
essential for protein–protein interactions with accessory factors, such as
PCNA (see below). The corresponding C-terminal domain in human Pol
is much longer (280 vs. 120 amino acids [aa]).
The crystal structure of Pol was determined in the absence of DNA, but
the location of DNA within the polymerase was determined by fitting the
crystal structure of Pol to the previously reported template-primer-
ddNTP ternary complex of T7 DNA polymerase (Trincao et al., 2001).
Using such an approach, it was shown that the binding pocket of the
polymerase around the templating base is likely to be open or extended,
thereby providing an explanation for the low fidelity of the polymerase
and suggesting a mechanism by which altered bases in the template can be
accommodated within the active site. In particular, modeling of a cova-

lently linked thymine dimer into the active site of Pol suggests it has
the capacity to accommodate two templating residues. Support for this
model comes from recent structural studies of Dpo4, a Y-family polymerase
from Sulfolobus solfataricus, whose structure has been solved in a ternary
complex with CDP-containing DNA and an incoming nucleotide (Ling
et al., 2003). The Dpo4-CPD structure clearly showed that the covalently
linked CPD was readily accommodated within the active site of Dpo4.
Modeling of Pol onto the Dpo4 structure further suggested that
the finger, thumb, and LF polymerase domains undergo a conformational
change from an ‘‘open,’’ DNA-free complex to a ‘‘closed,’’ CPD-
containing complex, which facilitates efficient bypass of the CPD (Ling
et al., 2003).
The possibility that Pol retains both thymine bases of the CPD within
its active site and directs incorporation of the correct nucleotides by using
the intrinsic base-pairing ability of the lesion is also supported by kinetic
studies (Washington et al., 2003b). Furthermore, structural and biochemi-
cal data suggest that intact Watson–Crick base pairing rather than the
geometric fit of the incoming nucleotide with the templating base within
the polymerase active site plays a crucial role in the efficiency and fidelity
of DNA synthesis by Pol. Thus, Pol accommodates a CPD lesion because
of the openness and flexibility of the active site, but also because Watson–
Crick base pairing, although weakened, remains intact at the CPD. Simi-
larly, yeast Pol readily traverses through 8-oxoG with the preferential
formation of a C:8-oxoG base pair, which retains the three hydrogen bonds
found in a normal C:G base pair (Haracska et al., 2000). In contrast to the
CPD and 8-oxoG, an abasic site or 6-4PP lesion represents a strong obstacle
to replication by Pol, as any hydrogen bonding potential is completely
absent at an abasic site and is significantly disrupted at the 30 side of the
6-4PP. The importance of the ability to form normal hydrogen bonds during
Pol-catalyzed DNA synthesis is further supported by the drastic decrease in
insertion fidelity of yeast Pol in replication reactions with difluorotoluene,
a nonpolar isosteric analog of thymine that is unable to form Watson–Crick
hydrogen bonds with adenine but is virtually identical to dT in shape, size,
and conformation (Washington et al., 2003a).
VI. REGULATION AND LOCALIZATION OF POL AND POL

The human POLH gene is expressed in all tissues, with small increases in
expression in proliferating tissues such as testis, thymus, and skin, and also
in the liver (Yamada et al., 2000). In mouse cells, Pol expression increased
when serum-starved cells were stimulated to proliferate (Yamada et al.,
216 VAISMAN ET AL.
2000). However, Pol is not required for normal DNA replication, as XP-V
cells proliferate normally in the absence of DNA damage (Lehmann et al.,
1975). Although the level of POLH mRNA decreases temporarily after
UV irradiation (Yamada et al., 2000), protein levels are unaffected
(P. Kannouche and ARL, unpublished observations). Both human and
murine POLI are similarly expressed in all tissues, with the highest level of
expression observed in postmeiotic spermatids from testis (Frank et al.,
2001; McDonald et al., 1999).
To gain insight into how Pol and Pol function inside the cell, localiza-
tion studies have been carried out using eGFP-tagged polymerase con-
structs (Kannouche et al., 2001, 2002). This work showed that both
polymerases were localized exclusively in the nucleus and were uniformly
distributed through the nucleus in the majority of cells in an asynchro-
nous culture of SV40-transformed human fibroblasts. However, in a small
proportion of the cells (typically 15%), the polymerases accumulated in
many bright foci, where they colocalized both with each other and with
PCNA. These foci are thought to be replication factories, in which DNA
replication is occurring. After treatment with UV irradiation, the number
of cells containing bright foci of colocalizing Pol, Pol, and PCNA
increased up to about 70% of the population (Fig. 3). It is believed that
the number of foci increases because UV-induced DNA damage blocks
progression of the replication forks and slows down cell passage through S
phase. The resulting gradual accumulation of cells in S phase accounts for
the increase in the number of cells with foci containing Pol, Pol, and
PCNA, rather than DNA damage-induced relocalization of these proteins,
as it was initially postulated. Consistent with this idea, accumulation of
nuclei with foci containing Pol and Pol has also been observed after
FIG. 3. Colocalization of Pols and in response to UV irradiation. Foci formation

after UV damage is shown for Pol (red) (left) and Pol (green) (middle). Both
polymerases form foci in the same time and space within a cell following UV irradiation,
indicating that the accumulation of Pol and Pol at replication forks stalled at sites of
UV damage is coordinated in vivo. The colocalization of Pol and Pol is indicated by a
yellow pattern in the merged figure (right). This figure is reproduced with permission
from Kannouche et al., 2002. (See Color Insert.)
other treatments that block progression of the replication fork, such as

methyl methanesulfonate or N-acetoxy-acetylaminofluorene, which gener-
ate DNA damage, and hydroxyurea, which arrests cells in S phase, without
causing DNA damage. The localization of Pol in replication foci is partly
dependent on the presence of Pol because in XP-V cells, Pol remains
nuclear, but its accumulation in replication foci is reduced approximately
three- to six-fold (Kannouche et al., 2002).
Deletion analysis has identified the domains required for correct locali-
zation of the polymerases (Fig. 2A). For Pol, the last 70 aa, containing a
bipartite nuclear localization signal, are sufficient for nuclear localization,
but not for accumulation into foci. However, the last 120 aa are sufficient
for localization both in the nucleus and in nuclear foci (Kannouche et al.,
2001). This 120-aa region contains a C2H2 zinc finger motif and, at the
extreme C terminus, a PCNA-binding motif (Fig. 2A). Both of these are
required for localization into foci (P. Kannouche and ARL, unpublished
results). Pol does not contain any obvious motifs in the unconserved C-
terminal part of the protein, and similar analyses were much less clear-cut.
Localization into the nucleus required sequences contained within aa
219–451 at the end of the polymerase domain and the beginning of the
nonconserved domain, whereas foci formation required the C-terminal
220 aa (Fig. 2A; Kannouche et al., 2002). Pol and Pol were shown to
interact directly, and this same C-terminal 220 aa of Pol were required for
the interaction with Pol. Taken together with the partial dependence on
Pol for foci formation, these data suggest that Pol helps transport Pol
into nuclear foci.
The cellular role of aa 430–600 of Pol, which is poorly conserved
among the Pol orthologs, is currently unknown.
VII. MUTATIONS IN POL IN XP VARIANTS

The two initial studies in which POLH was discovered to be the gene
defective in XP-V cells also identified several nonsense mutations in
human patients with XP-V ( Johnson et al., 1999a; Masutani et al., 1999).
Subsequent work identified many further mutations in XP-V patients
(Broughton et al., 2002; Itoh et al., 2000), and these are summarized in
Figure 4. The majority of the mutations cause truncations in Pol that
are located within the conserved polymerase domain found in all Y-family
polymerases and that are likely to produce a completely inactive protein.
This suggests that Pol is not required for human life. A few truncat-
ing mutations are located close to the C terminus, outside of the polymer-
ase domain. Extracts from these cells have TLS activity characteristic for
Pol (Broughton et al., 2002), but we anticipate that the truncated Pol
218
VAISMAN ET AL.
FIG. 4. Locations of mutations in POLH that lead to the Xeroderma pigmentosum variant (XP-V) phenotype. The locations of the amino
acid changes identified in patients with XP-V are shown relative to the different domains of the polymerase shown in Fig. 2B. The structural
domains of Pol are shown in blue (finger, F), red (palm, P), green (thumb, T), and purple (little finger, LF) in the central part of the diagram.
The nuclear localization signal (NLS) and the domain involved in localizing Pol into replication foci (URS) are also indicated in black and
brown, respectively. Cell strains are designated in boxes. Subscripts 1 and 2 specify different alleles. Deletions are shown using horizontal lines.
Single amino acid changes are shown in the upper part of the diagram, and truncations are shown in the lower part. (See Color Insert.)
molecules would probably not reside in the replisomes, as they are missing
the C-terminal domain required for localization in the nucleus and in
nuclear foci.
Several missense mutations, mostly located within the conserved catalyt-
ic domain, have also been identified (Fig. 4, top). These were modeled
onto the three-dimensional structure of Dpo4 from Sulfolobus solfataricus.
Two of the mutated amino acids, G263 and R361, are involved in interac-
tions with the DNA, whereas the other mutations are likely to disrupt the
conformations of the different domains (Broughton et al., 2002). Interest-
ingly, a Japanese patient is a compound heterozygote for two mutations,
K535E and K589T, both located in the poorly conserved central domain
between the polymerase and localization domains (Itoh et al., 2000). K535
is, however, in a run of nine aa conserved in mammalian species of Pol,
and K589 is conserved in mouse and rat, demonstrating important
functions for this part of the protein.
As mentioned above, the severely truncating mutations suggest that Pol
is not an essential gene in humans, and in support of this idea, a trans-
genic mouse lacking Pol has recently been generated (Fumio Hanaoka,
personal communication).
VIII. POLS AND AND THE POLYMERASE SWITCH: INTERACTIONS WITH

PCNA AND REV1
A topic of major interest that is also discussed in more detail in other
chapters of this book is the mechanism by which the replicative DNA
polymerase is displaced at a replication fork blocked by DNA damage and
is replaced with a TLS polymerase. All three of the E. coli TLS polymerases
have motifs involved in the interaction with the sliding clamp that helps
to recruit polymerases to their site of action (Becherel et al., 2002). In
eukaryotic cells, proliferating cell nuclear antigen (PCNA) plays the role
of a sliding clamp, and analyses of the primary aa sequences of Pols and
reveal putative PCNA-binding motifs. Yeast two-hybrid assays confirmed
that there is a physical interaction between Pol and PCNA in vivo
(Haracska et al., 2001a); and in vitro replication assays revealed that the
catalytic activities of both Pol and Pol on undamaged and damaged DNA
templates were increased by the presence of PCNA along with clamp
loader, replication factor C (RFC), and RPA (replication factor A)
(Haracska et al., 2001a,b).
As discussed in detail in Chapter 10, PCNA becomes both mono- and
polyubiquitinated in yeast cells following treatment with MMS (Hoege
et al., 2002). Based on these findings, it was postulated that PCNA modifi-
cation was involved in the polymerase switch (Hoege et al., 2002), and this
220 VAISMAN ET AL.
idea was supported by genetic evidence showing that ubiquitination of

PCNA was epistatic with POL30 (Stelter and Ulrich, 2003). In mammalian
cells, in response to UV irradiation, PCNA is only monoubiquitinated, and
this ubiquitination is not dependent on NER or on Pol. The monoubi-
quitinated PCNA is found exclusively in chromatin, where it interacts
physically and specifically with Pol (Kannouche et al., 2004). This inter-
action, detected by coimmunoprecipitation, was found only with the
modified form of PCNA. No interaction was detected with unmodified
PCNA in cell extracts. The increased affinity for Pol of monoubiquiti-
nated PCNA generated at blocked replication forks therefore provides an
attractive mechanism for mediating the polymerase switch at the sites of
DNA damage.
As discussed at the beginning of this chapter, Rev1 is phylogenetically
related to the Y-family polymerases, but it primarily inserts dCMP, and
not the three remaining dNTPs. Recently, several groups have inde-
pendently demonstrated that Pol aa residues 370–492 interact with the
C-terminal 100 aa of Rev1 (Fig. 2A) (Guo et al., 2003; Ohashi et al., 2004;
A. Tissier et al., 2004). The same C-terminal fragment of Rev1 also inter-
acts with Pol, Pol (see Chapter 9), and Rev7 (see Chapter 6) (Guo et al.,
2003; Ohashi et al., 2004; Tissier et al., 2004). Revl, like Pols and , is
localized in replication factories (Tissier et al., 2004). These findings
suggest that Revl might play a role as a molecular scaffold during the
bypass of DNA lesions, facilitating aspects of the polymerase switching
mechanisms.
IX. PROTECTION FROM CELLULAR EFFECTS OF DNA DAMAGE

Early work showed that, in contrast to NER-defective XP cells, XP-V
fibroblasts were only marginally sensitive to killing by UV light (although
they are specifically sensitized by caffeine). They were, however, like NER-
deficient XP cells, highly sensitive to the induction of mutations by UV
light (Maher et al., 1976). We may now interpret these data as suggesting
that TLS by Pol plays only a minor role in protecting human cells from
killing by UV light but has a major role in safeguarding us from mutations.
This is absolutely consistent with the finding that Pol in most instances
inserts the ‘‘correct’’ nucleotides opposite CPDs (discussed above, in
section III), and that in its absence, the substituting mechanism is much
more error prone. In recent work supporting these findings, cell lines
have been derived in which XP-V cells stably express a transfected POLH
gene. UV mutagenesis was measured using two different systems, and the
mutant frequency was restored to normal levels in the Pol-transfected
cells (Stary et al., 2003; Taylor et al., 2003).
The UV mutation spectra in XP-V cells differ significantly from those in

normal or NER-defective XP cells. In particular, whereas most UV-induced
mutations in the latter two cell types are C to T transitions, in XP-V cells there
is a greater proportion of mutations at thymines (Wang et al., 1993). Thus, in
the absence of Pol, the substituting enzymes make more errors at A or T
residues. On the basis of biochemical data, it seems reasonable to hypothe-
size that Pol might be the enzyme responsible for such phenotype. Although
the ability of Pol to replicate CPD-containing templates is much lower than
that of Pol, it is higher than that of any other known eukaryotic polymerase
and is very similar to that observed for the well-characterized E. coli transle-
sion polymerase, PolV (Tang et al., 2000; Vaisman et al., 2002). In addition,
the highly error-prone behavior of Pol at template T (Tissier et al., 2000a),
and the increased accuracy of nucleotide incorporation opposite deami-
nated cytosines compared to other polymerases (Vaisman and Woodgate,
2001), is consistent with the mutation spectra observed in XP-V cells.
X. ROLES OF POL AND POL IN SOMATIC HYPERMUTATION

Somatic hypermutation (SHM) is one of the mechanisms by which
antibody diversity is generated and is the subject of Chapter 11. Models
for the mechanism of SHM implicate an error-prone polymerase, and
several groups have investigated the involvement of different Y-family
polymerases in this process. In lymphocytes from three XP-V patients,
the frequency of SHM was normal but the types of base changes were
different: There was a decrease in mutations at A and T and a concomitant
rise in mutations at G and C. These results suggest that more than one
polymerase contributes to SHM, and that if one is absent, others compen-
sate. The data suggest that Pol is involved in generating errors that occur
predominantly at A and T and that another polymerase may preferentially
generate errors opposite G and C (Zeng et al., 2001). In support of this
idea, the SHM hotspots at A and T residues correlate with the Pol error
spectrum (Rogozin et al., 2001). With regard to Pol, the data presently
available are inconclusive. Targeted inactivation of the POLI gene in the
Burkitt’s lymphoma BL2 cell line abolished SHM, suggesting that Pol is
absolutely required for SHM in these cells (Faili et al., 2002). In contrast,
the frequency and spectrum of SHM were indistinguishable in a
129-derived mouse strain that contains a stop codon early in the Poli gene
and in Polþ mice (McDonald et al., 2003). These latter data suggest that
Pol is dispensable for SHM. However, both systems have their peculia-
rities, and so extrapolation to the human population must be treated with
caution. It therefore remains plausible that Pol, like Pol, may play a role
in SHM, but that the various polymerases have redundant functions.
222 VAISMAN ET AL.
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PROPERTIES AND FUNCTIONS OF ESCHERICHIA COLI:
POL IV AND POL V
By ROBERT P. FUCHS, SHINGO FUJII, AND JÉRÔME WAGNER
Cancérogenèse et Mutagenèse Moléculaire et Structurale,

CNRS ESBS, 67400 Strasbourg, France
I. DNA Pol IV, the dinB Gene Product . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 230

A. Discovery of the dinB Gene Product Activity . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 230
B. Biochemical Properties of DinB/Pol IV. . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 232
C. In Vivo Functions of Pol IV . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 239
D. Regulating the Access of Pol IV to Replication Intermediates . . . . . . . . . . .. . . . . . 242
II. DNA Polymerase V, the umuDC Gene Product . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 248
A. Genetic Requirements of Induced Mutagenesis . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 248
B. Biochemical Properties of Pol V . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 250
C. Switches Between Replicative and Specialized DNA Polymerases
During Lesion Bypass . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 255
References . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 257
Abstract
Escherichia coli possesses two members of the newly discovered class of Y
DNA polymerases (Ohmori et al., 2001): Pol IV (dinB) and Pol V (umuD 0 C).
Polymerases that belong to this family are often referred to as specialized
or error-prone DNA polymerases to distinguish them from the previously
discovered DNA polymerases (Pol I, II, and III) that are essentially involved
in DNA replication or error-free DNA repair. Y-family DNA polymerases
are characterized by their capacity to replicate DNA, through chemically
damaged template bases, or to elongate mismatched primer termini.
These properties stem from their capacity to accommodate and use dis-
torted primer templates within their active site and from the lack of an
associated exonuclease activity. Even though both belong to the Y-family,
Pol IV and Pol V appear to perform distinct physiological functions.
Although Pol V is clearly the major lesion bypass polymerase involved in
damage-induced mutagenesis, the role of Pol IV remains enigmatic. In-
deed, compared to a wild-type strain, a dinB mutant exhibits no clear
phenotype with respect to survival or mutagenesis following treatment with
DNA-damaging agents. Subtler dinB phenotypes will be discussed below.
Moreover, despite the fact that both dinB and umuDC loci are controlled
by the SOS response, their constitutive and induced levels of expression
are dramatically different. In noninduced cells, Pol V is undetectable by
Western analysis. In contrast, it is estimated that there are about 250 copies

230 FUCHS ET AL.
of Pol IV per cell. On SOS induction, it is believed that only about 15

molecules of Pol V are assembled per cell (S. Sommer, personal communi-
cation), whereas Pol IV levels reach 2500 molecules. In fact, despite
extensive knowledge of the individual enzymatic properties of all five
E. coli DNA polymerases, much more work is needed to understand how
their activities are orchestrated within a living cell.
I. DNA POL IV, THE DINB GENE PRODUCT
A. Discovery of the dinB Gene Product Activity

The dinB (din stands for DNA damage inducible) locus was origi-
nally described by Kenyon and Walker in the early 1980s (Kenyon and
Walker, 1980) when searching for E. coli genes whose expression is up-
regulated by DNA damage. Some 6 years later, Brotcorne-Lannoye and
Maenhaut-Michel (1986) found the first dinB phenotype; namely, the
inability of an E. coli strain carrying the original Mu d1(Ap Lac)–dinB
fusion (isolated by Kenyon and Walker, 1980), which disrupted the dinB
locus, to promote untargeted mutagenesis of bacteriophage . Untargeted
mutagenesis is defined as mutations occurring in undamaged lambda
phage as a result of being replicated within an ultraviolet (UV)-irradiated
host cell. This pathway was found to be independent of UmuD0 C and
RecA, the key players involved in induced chromosomal mutagenesis.
Almost 10 years later, as part of the E. coli genome sequencing project
in Japan, Ohmori et al. (1995) identified an ORF that they called dinP, with
a putative LexA (the repressor of the SOS response) binding site within its
promoter region. The 1056 bp long dinP ORF was subsequently shown to be
identical to the dinB locus and encodes for a 351–amino acid, rather basic
protein with a calculated pI and molecular weight of 9.4 and 39,516 Da,
respectively (Kim et al., 1997). These authors further showed that disrup-
tion of the dinP gene abolishes the untargeted mutagenesis of phage and
that overexpression of DinB dramatically increases the spontaneous muta-
tion frequency in E. coli cells (hereafter designated the DinB mutator
phenotype).
Sequence analyses reveal that the dinB gene product shares strong local
sequence homologies with UmuC-like proteins, which include Saccharomy-
ces cerevisiae REV1 protein (Kulaeva et al., 1996; Larimer et al., 1989;
Ohmori et al., 1995). Multiple sequence alignment of the UmuC-like
proteins revealed five conserved motifs. Figure 1 shows the alignment of
motifs I–V (Kulaeva et al., 1996) from six representative members of the
UmuC-like proteins.
POL IV AND POL V IN E. COLI
FIG. 1. The five conserved domains in the DinB/UmuC/Rev1-like proteins. Multiple alignment (Clustal W; Thompson et al., 1994) of
the Escherichia coli (eubacterial) DinB (Pol IV) and UmuC (Pol V), Sulfolobus solfataricus and Sulfolobus acidocaldarius (archeal) Dpo4 and
Dbh, Saccharomyces cerevisiae (lower eukaryote), Rev1 and Homo sapiens (higher eukaryote) RAD30 A (XPV or Pol ) shows five conserved
motifs in all these proteins. Arrows mark specific amino acids discussed within the text. Numbers indicate the distances (in amino acids)
separating the conserved domains and the distances from the proteins termini. The bottom line (conservation) indicates the strictly
conserved residues, two dots (:) indicate full conservation among groups of strongly related amino-acids (STA, NEQK, NHQK, NDEQ,
QHRK, MILV, MILF, HY, or FYW); one dot (.) indicates conservation among groups of weakly related amino-acids (CSA, ATV, SAG,
STNK, STPA, SGND, SNDEQK, NDEQHK, NEQHRK, FVLIM, or HFY).
231
232 FUCHS ET AL.
Kulaeva et al. (1996) also noted that ‘‘the most conserved portion of
motif III, with adjacent invariant aspartic acid and glutamic acid residues
preceded by a stretch of hydrophobic residues, resembled the Mg2þ-bind-
ing site that is conserved in a variety of ATPases’’ and that ‘‘the high level of
sequence conservation between UmuC-like proteins from bacteria, archaea
and eukaryotes suggests that these proteins may have an enzymatic activity,
the nature of which remains to be determined’’ (pp. 250–251). This hypoth-
esis was shown to be true later the same year when C. Lawrence and cow-
orkers showed that the REV1 protein was endowed with a highly specific
deoxycitidyl transferase activity in vitro (Nelson et al., 1996a). These two
studies therefore provided the first hint that all members of this family
might similarly possess a nucleotidyl transferase activity. Moreover, al-
though no direct homology between UmuC/DinB/REV1 family members
and known nucleotidyl transferases could be found at the primary sequence
level, the few strictly conserved acidic residues in motifs I and III, as well
as the positively charged residue (R or K) in motif II, are actually the same as
those found in conserved motifs A, B, and C of known DNA polymerases
(Delarue et al., 1990). Altogether, such observations provided the first
clues as to the true biochemical functions of the DinB/UmuC-like proteins;
namely, that they are DNA polymerases. As discussed in the following
sections of this chapter, such activity was demonstrated not only for both
dinB and umuC gene products of E. coli but also for other homologues from
all three domains of life (see Chapter 6 by Lawrence; Chapter 7 by Vaisman,
Lehmann, and Woodgate; and Chapter 9 by Ohashi, Ohmori, and Ogi).
These polymerases form the so-called Y family of DNA polymerases and are
sometimes referred to as error-prone, specialized, or TLS (for translesion
synthesis) polymerases. Although these specialized DNA polymerases are
clearly involved in the replication of damaged templates and are, as such,
responsible for a high proportion of induced mutations, it should be
kept in mind that some lesions can be replicated by the so-called replicative
DNA polymerases, thus causing mutations as well (see later discussion).
B. Biochemical Properties of DinB/Pol IV
1. dinB Gene Encodes a Bona Fide Template-Directed DNA Polymerase,

DNA Pol IV
When the catalytic activity of a highly purified HisTag-DinB fusion
protein was directly tested in the laboratory of T. Nohmi (NIHS, Tokyo)
in 1998, it was found to possess a bona fide template-directed DNA poly-
merase activity (Fig. 2; Wagner et al., 1999), rather than a highly specific
nucleotidyl transferase, as observed previously for REV1 (Nelson et al.,
POL IV AND POL V IN E. COLI 233
FIG. 2. The dinB gene encodes a bona fide template-directed DNA polymerase. The
specificity of nucleotide incorporation by highly purified histidine-tagged DinB was
investigated by reacting 30 nM of the specified substrates with 10 nM enzyme, with or

without dNTP (125 M), as indicated for 10 minutes at 37 C. (Adapted from Wagner
et al., 1999, with permission.)
1996) and Mgþþ ions were found to be essential for DNA polymerase
activity. As noted above, primary sequence analysis of DinB and its homo-
log identified conserved residues potentially critical for the catalytic activi-
ty of the protein. Specifically, Asp8, Asp103, and Glu104 may correspond
to the three acidic residues of ‘‘classic’’ DNA polymerases necessary to
coordinate the divalent cations engaged in catalysis. Mutating any of these
residues abolished the catalytic activity of this protein in vitro (Wagner et al.,
1999), as well as the previously documented DinB mutator phenotype
in vivo (Kim et al., 1997), thus directly linking the polymerase activity of
DinB with its mutagenic properties. Likewise, mutating the highly con-
served Arg49 in motif II severely compromised the activity of DinB
(Wagner et al., 1999). Since then, structural studies performed on eukary-
otic and prokaryotic homologs of DinB confirmed the critical roles of the
three acidic residues in motifs I and III that are coordinating the divalent
cations and of the positively charged residue in motif II that interacts with
the incoming dNTP (Boudsocq et al., 2002; Ling et al., 2001; Silvian et al.,
2001; Trincao et al., 2001; Zhou et al., 2001). Similarly, a highly conserved
aromatic residue in Y polymerases (F13 and F12 in Pol IV of E. coli and in
Dbh of Sulfolobus acidocaldarius [Boudsocq et al., 2004], respectively) allows
discrimination against the incorporation of ribonucleotides (DeLucia et al.,
2003; Shimizu et al., 2003; Zhou et al., 2001).
Pre-steady-state studies of Y-family polymerases indicate that for nu-
cleotidyl transfer reaction to occur, the polymerases have to undergo
234 FUCHS ET AL.
conformational changes before the chemical step (Fiala and Suo, 2004b;
Washington et al., 2001). It was thus concluded that Y-family polymerases
perform a fidelity check, involving an ‘‘induced-fit’’ mechanism. The
classic induced-fit mechanism is defined as the finger subdomain under-
going a large conformational change from an ‘‘open’’ state to a ‘‘closed’’
state on binding of a correct incoming nucleotide to the polymerase
(Johnson, 1993). If the incoming nucleotide does not match the template
base, the proper ‘‘closed’’ state cannot be attained, which in turn prevents
the chemical reaction. However, crystal structures of the Y-family poly-
merases, including two archaeal members (Dbh and Dpo4) and yeast
Pol , determined to date indicate different conformational changes
(see review by Yang, 2003). The catalytic core of the two closely related
Y polymerases, Dbh and Dpo4, is virtually superimposable, despite the fact
that Dbh is free of substrates and Dpo4 is complexed with DNA and a
correct incoming nucleotide. Comparison of the Dbh apoprotein struc-
ture with the replicative DNA polymerases indicates that Dbh is already in
the ‘‘closed’’ state, even without any substrate (Zhou et al., 2001). Struc-
tural changes of Dpo4 on association with DNA do occur, as the crystal
structures reveal (Ling et al., 2004), but the domain that moves is not the
finger subdomain but, rather, the ‘‘little finger,’’ far away from the
replicating base pair. Even though the finger subdomain of Pol may
undergo some conformational changes (Ling et al., 2003), the largest
movement still occurs with the little finger. Therefore, the conformational
step observed in Dpo4 and Pol by pre-steady-state kinetic analyses may
differ from the classic ‘‘induced-fit’’ mechanism.
2. Processivity of Pol IV and Modulation of Its Activity Through
Interaction with the Clamp
When examined in a classical primer-extension assay using a simple
primer-template DNA substrate, Pol IV produces a ladder-like pattern
(see Fig. 2) illustrating its poor processivity. In fact, Pol IV appears to
be strictly distributive, catalyzing the incorporation of only 1 nucleotide
per binding event (Wagner et al., 1999). This feature may be related to
an extremely low affinity of Pol IV for the 30 -OH extremity of a naked primer
template substrate (Gruz et al., 2001; Wagner et al., 2000). The fact that
no stable complex between DNA and Pol IV has been observed indicates that
accessory factors allow the formation of a more stable polymerase/DNA
substrate complex (Wagner et al., 2000). The clamp, which is also known
as the processivity subunit of the replicative DNA polymerase (Pol III),
fulfills this role. Once loaded onto DNA by the multisubunit clamp loader,
complex, the clamp confers the requested high processivity to the
replicative DNA polymerase. It turns out that this sliding clamp dramatically
alters the activity of Pol IV, increasing its affinity for the substrate, synthesis
efficiency, and apparent processivity by two to four order of magnitude
(Table I; Tang et al., 2000; Wagner et al., 2000).
The dissociation rate of the Pol IV--DNA complex has been determined
to be 0.005 sec1, which corresponds to a half-life of the complex of about
140 seconds. Thus, with a measured kpol of 2 nucleotides per second, the-
calculated processivity of Pol IV- is 300–400 nucleotides. As discussed later
in this chapter, the interaction between Pol IV and is essential for its
role in mutagenesis. However, whether Pol IV synthesizes hundreds of
nucleotide-long tracks in vivo remains to be determined.
3. Fidelity of Pol IV
As opposed to classical E. coli DNA polymerases Pol I, Pol II, and Pol III
core, Pol IV is devoid of any intrinsic 30 to 50 exonuclease (proofreading)
activity (Wagner et al., 1999). This implies that the Pol IV fidelity is
achieved solely by its capacity to discriminate between correct and incor-
rect base-pair formation. As shown in Fig. 2, Pol IV preferentially catalyses
the incorporation of the correct nucleotides opposite all four template
bases.
Actually, steady-state kinetic analysis of the misinsertion capacities of
Pol IV opposite all four template bases showed that Pol IV is, on average,
only four- to fivefold less accurate than the catalytic -subunit of
the replicative Pol III (Kobayashi et al., 2002; Tang et al., 2000). This obser-
vation suggests that, despite dinB being associated with mutagenesis, Pol IV
is, at least at the incorporation step, a relatively faithful enzyme. Recent
pre-steady-state kinetic studies of S. solfataricus DNA Pol IV (Dpo4) show that
TABLE I
Modifications of Kinetic Parameters of Pol IV on Interaction with
Pol IV Pol IV þ Fold stimulation
koff (S1) n.d. 0.005a n.a.

kpol (S1) n.d. 2a n.a.
Processivity (nucleotides) 1a 400a 400
KmDNA (nM) 7180b 0.2a 35 900
KmdNTP (M) >2 000c 0.12a >16 000
Vmax (min1) n.d. 0.3a n.a.
Vmax/Km (M1 min1) 1.1 103c 2.5a 2 270
a
Data from Wagner et al. (2000).
b
Data from Gruz et al. (2001).
c
Data from Tang et al. (2000).
(n.d.) not determined, (n.a.) not available.
236 FUCHS ET AL.
the low fidelity of this enzyme mainly results from a weak discrimination
between the correct and incorrect incoming nucleotide at the initial nucle-
otide binding step (Fiala and Suo, 2004b). This property is related to the
observed solvent accessible and open structure of the Dpo4 active site (Ling
et al., 2001). Another aspect of DNA polymerase low fidelity resides in the
capacity to elongate mismatched primer/template termini. Although Pol IV
is able to extend mismatches, it is less efficient than its human counterpart
Pol (Kobayashi et al., 2002). However, as discussed next, efficient extension
of mismatches by Pol IV and its homologs may be sequence context specific.
Thus, it is worth noting that although Y-family DNA polymerases are gener-
ally considered as ‘‘error-prone’’ enzymes, huge differences in their specific
properties are observed.
The efficiencies of Pol IV in incorporation and extension of each
mismatched base pair are summarized in Fig. 3 (Kobayashi et al., 2002),
where the mismatches appearing in the upper-right corner of the panel
are most efficiently generated and extended. These data are in relatively
FIG. 3. In vitro fidelity of Pol IV. Nucleotide misinsertion efficiencies (fins) are
plotted versus mismatch extension efficiencies (fext). Data points above the line indicate
lower values for the misinsertion efficiency compared to mismatch extension; inversely,
data falling below the line indicate higher values for misinsertion compared to
mismatch extension. (Reproduced from Kobayashi et al., 2002, with permission.)
good agreement with the mutation spectra determined in vitro or in vivo,

excepting T to A transversions that are not observed in vivo (Kobayashi
et al., 2002; Wagner and Nohmi, 2000).
The dinB-dependent untargeted mutagenesis pathway, as well as the DinB
mutator activity, are associated with a specific increase in 1 frameshift
mutagenesis within homopolymeric runs of 6 and more G:C base pairs
(Kim et al., 1997; Wood and Hutchinson, 1984). The capacity of Pol IV to
elongate frameshift intermediates was also confirmed in vitro (Wagner et al.,
1999). However, analysis of forward mutational spectra obtained either
in vivo or in vitro highlighted other specificities of Pol IV–induced mutagen-
esis. First, 1 frameshifts are strongly induced within both G:C and A:T
single-nucleotide repeats, and more important, short repeats and nonrepe-
titive sequences are also susceptible to deletion events catalyzed by Pol IV
(Kobayashi et al., 2002; Wagner and Nohmi, 2000). Second, base substitu-
tions are also strongly induced by PolIV, representing 18%–30% of all
mutagenic events observed in vitro and in vivo, respectively (Kobayashi
et al., 2002; Wagner and Nohmi, 2000). Remarkably, changes toward G:C
pairs represent 70% of all Pol IV–induced base substitutions. The mutation-
al spectrum obtained in vivo by overexpression of Pol IV in E. coli indicates
that a vast majority of both base substitutions toward G:C pairs as well as 1
frameshifts occur within a 50 -GX sequence context, in which X represents
the deleted or mutated base (Wagner and Nohmi, 2000). Actually, kinetic
and structural studies strongly support the notion that Pol IV and homologs
such as the S. solfataricus Dpo4 and S. acidocaldarius Dbh polymerases may be
particularly prone to ‘‘dNTP-stabilized misalignment’’ (Fig. 4) (Fiala and
Suo, 2004a; Kobayashi et al., 2002; Kokoska et al., 2002; Potapova et al., 2002).
Ling et al. (2001) described a crystal structure of a Dpo4 ternary complex that
accommodates two undamaged template bases in its active site, with the
incoming nucleotide paired with the second (50 ) template base. Recently,
the same group reported a crystal structure of Dpo4 complexed with an
abasic lesion, in which Dpo4 loops out the abasic site and uses the base 50 to
the lesion to direct nucleotide incorporation (Ling et al., 2004). These
structures illustrate two alternative mechanisms to generate a 1 frameshift
invoked in the ‘‘dNTP stabilized misalignment’’ model presented in Fig. 4.
Why this specific sequence context (50 -GX) represents a ‘‘hot spot’’ for
mutagenesis induced by Pol IV remains to be determined.
4. Involvement of Pol IV in Lesion-Induced Mutagenesis and Translesion

Synthesis In Vitro
Although the umuDC genes products (Pol V) were known to be required
for base substitution mutagenesis triggered by UV light or abasic sites
(review by Smith and Walker, 1998), the discovery of the Y family of DNA
238 FUCHS ET AL.
FIG. 4. A ‘‘dNTP-stabilized misalignment’’ model for the Pol IV polymerases

induced mutagenesis at 50 GX sequences. (a, b): An incoming dCTP residue is
noncomplementary to the template cytosine residue in bold but can form a Watson–
Crick base pair with the 50 adjacent guanosine, leaving the template cytosine unpaired.
Such an intermediate, which is referred to as a ‘‘dNTP-stabilized misalignment’’
intermediate, has been observed in a ternary complex in which an incoming dGTP was
unable to pair with the template G but could form a base pair with the 50 adjacent C
residue (Ling et al., 2001). If a phophodiester bond between the misaligned incoming
dNTP and the 30 -OH extremity of the primer can be formed (c), it can lead either to a
1 frameshift mutation (direct extension, pathway F) or to a base substitution after
realignment and mismatch extension (pathway B). (Reproduced from Kokoska et al.,
2002, with permission.)
polymerases led to the evaluation of the implication of other polymerases

in lesion-induced mutagenesis and TLS.
As far as E. coli Pol IV and archaeal Dbh and Dpo4 homologs are
concerned, several studies addressed this question through in vitro experi-
ments involving purified polymerases and, eventually, accessory proteins
such as the processivity clamp (Boudsocq et al., 2001; Gruz et al., 2001;
Kobayashi et al., 2002; Maor-Shoshani et al., 2003; Shen et al., 2002; Suzuki
et al., 2001; Tang et al., 2000). From these studies, it turns out that Pol IV
has the capacity to perform in vitro DNA synthesis across a wide panel of
base modifications [8-oxoguanine; O6-methylguanine, uracil, abasic sites,
N-2-acetylaminofluorene and N-2-aminofluorene modified guanines, cis-
syn cyclobutane dimers and 6-4 pyrimidine-pyrimidone TT photoproducts,
1,2-cisplatinated guanine adduct, and the major benzo[a]pyrene diol
epoxide-N2-guanine (B[a]P guanine) adduct] with various efficiencies.
In vitro bypass of abasic sites and B[a]P guanine adducts by Pol IV are
particularly efficient (Maor-Shoshani et al., 2003; Shen et al., 2002). How-
ever, although Pol IV is clearly involved in the bypass of a site-specific
B[a]P guanine adduct (see Section I.C.3 and Napolitano et al., 2000), it
does not seem to be involved in the in vivo bypass of a site-specific AP site
(Maor-Shoshani et al., 2003). The discrepancies between in vitro and in vivo
data, with respect to AP site bypass, strongly indicate that the activity of
these enzymes is regulated in vivo.
In addition to TLS, direct incorporation of modified nucleotides into
DNA constitutes a real threat to genome stability (Ames and Gold, 1991).
Interestingly, oxidized DNA precursors 8-OH-dGTP and 2-OH-dATP
are efficiently and erroneously incorporated into DNA by E. coli Pol IV
and its archaeal homologs, Dbh and Dpo4 (Shimizu et al., 2003; T. Nohmi,
personal communication). Actually, these polymerases preferentially incor-
porate 8-OH-dGTP opposite a template adenine, and 2-OH-dATP opposite
a template guanine. Moreover, these events do not prevent further elonga-
tion of the nascent chain by these polymerases. As concluded by Shimizu
et al. (2003), it turns out that Pol IV and homologs may promote mutagene-
sis through three distinct pathways that are spontaneous replication errors,
TLS, and misincorporation of modified dNTPs during DNA synthesis.
C. In Vivo Functions of Pol IV
1. In Vivo Expression Level of Pol IV

As noted above, the expression of the chromosomal dinB gene coding
for Pol IV is regulated as part of the SOS response. However, the LexA-box
located within the promoter region of dinB differs by 7 nucleotides from
the 20-nucleotide consensus sequence (Ohmori et al., 1995), possibly
explaining the high level of constitutive Pol IV expression (about 250
molecules per cell). Induction of the SOS response further increases its
expression about 10-fold, yielding
2500 molecules per bacteria (Kim
240 FUCHS ET AL.
et al., 2001). Moreover, Layton and Foster (2003) recently showed that the
general stress response sigma factor 38, encoded by the rpoS gene, also
participates in the regulation of the Pol IV intracellular level. The maximal
amount of Pol IV expressed from its single chromosomal locus actually
occurs in stationary phase. Remarkably, it is 30-fold higher than the
constitutive level, representing as much as 7500 Pol IV molecules per cell
(Layton and Foster, 2003). Thus, if one combines the high affinity of Pol
IV for -loaded DNA (see Section B.2. of this chapter) with its high cellular
concentration, it is tempting to speculate that Pol IV may be constitutively
part of the replisome, a notion that fits well with a previously suggested
role for Pol IV in assisting the replicative polymerase during synthesis of
particularly difficult sequence contexts (Wagner et al., 1999). It is difficult
to evaluate whether such a role for Pol IV is compatible with the lack of a
strong phenotype in dinB mutants.
2. Involvement of Pol IV in Long-Term Survival and Evolutionary
Fitness of E. coli
The high expression level of Pol IV indicates that there is an impor-
tant and basic function for Pol IV in general metabolism that remains,
however, to be discovered. In addition to the strict requirement for a
functional dinB gene in the untargeted mutagenesis of phage, a function
that provides no increase in bacterial fitness (Brotcorne-Lannoye and
Maenhaut-Michel, 1986), two other functions of Pol IV have been illu-
strated in vivo. One is the involvement of this polymerase in TLS of various
DNA lesions (see next paragraph), and the other is that the stationary
phase-dependent induction of Pol IV may correlate with the capacity of the
enzyme to enhance the long-term survival and evolutionary fitness of E. coli
(Yeiser et al., 2002). This property is also shared with the other two SOS-
induced DNA polymerases (Pol II and Pol V) and is characterized by the
inability of individual SOS polymerase mutant strains to maintain them-
selves when cocultured with wild-type cells during long-term stationary
phase incubation. Also, such mutant strains show partial defects in expres-
sing the ‘‘growth advantage in stationary phase’’ (GASP) phenotype. The
GASP phenotype, defined as the capacity of ‘‘aged’’ cells (that have
experienced a long stationary phase period) to take over a ‘‘young’’ cell
population depends on the appearance of new mutations that confer such
a competitive advantage. Because the culture conditions used in the GASP
experiments mimic long periods of nutrient stress that bacteria exper-
ience in natural environments (Morita, 1993), such phenotypes may, in
fact, be physiologically relevant. The fact that high expression levels of
Pol IV substantially increase spontaneous mutagenesis in both growing
cells (untargeted mutagenesis) and nonproliferating cells under nonlethal
selection (stationary phase or ‘‘adaptive’’ mutagenesis; (Kim et al., 1997;

McKenzie et al., 2001; Slechta et al., 2003; Strauss et al., 2000; Wagner
and Nohmi, 2000) fits well with the suggestion that Pol IV and homologs
may contribute to adaptive strategies of bacteria, including pathogens
(McKenzie and Rosenberg, 2001).
3. Pol IV–Dependent Translesion Synthesis and Mutagenesis In Vivo

In E. coli, the expression of Pol II, a proofreading-proficient B-family DNA
polymerase, and Pols IV and V, the two Y-family polymerases, is stimulated
through the induction of the SOS response identified in this bacterium.
All three polymerases are thought to participate in TLS in vivo (Napolitano
et al., 2000). In particular, Pol IV participates in TLS and mutagenesis
induced by B[a]P-guanine adduct, 4-nitroquinoline N-oxyde (4-NQO)–in-
duced DNA lesions and oxidative DNA damages (Kim et al., 2001; Lenne-
Samuel et al., 2000; Napolitano et al., 2000; Shen et al., 2002; Wagner et al.,
2002). It should also be noted that lesion bypass is by no means restricted to
Y-family DNA polymerases: Both replicative and specialized DNA poly-
merases allow cells to deal with the large diversity of ‘‘lesion/sequence
contexts’’ situations that are encountered by the replication fork (Table II).
This complexity is particularly well illustrated by the bypass of a site-
specific B[a]P-guanine adducts in E. coli. Within the GGA sequence context
(the adducted base shown in bold), both ‘‘error-free’’ and 1 frameshift
bypass pathways depend on both Pol IV and Pol V, whereas the G to T
TABLE II
All Three SOS Polymerases are Involved in TLS
Inducible polymerases involved in
Lesion Sequence context Error-free TLS Mutagenic TLS (mutation type)
T(6–4)a 50 -AATT- Pol V Pol V (T to C)

BPDEa 50 -GGG- Pol V þ Pol IV Pol V þ Pol IV (1)
AAFa 50 -GGG- Pol V Pol V (1)
AAFa 50 -gGCGCc- Pol V Pol II (2)
AAFb 50 -gGCGCGCc- n.d. Pol II or Pol V (2)
Oxydative 50 -gGCGCc- n.d. Pol V þ Pol IV (2)
lesionb
Oxydative 50 -gGCGCGCc- n.d. Pol V þ [Pol IV or II] (2)
lesionb
a
Data obtained using site specifically mono-modified plasmid DNA.
b
Data obtained using randomly modified plasmid DNA.
(n.d.) not determined. (Adapted from Wagner et al., 2002, with permission.)
242 FUCHS ET AL.
transversion pathway requires neither of them and is most probably per-

formed by Pol III (Lenne-Samuel et al., 2000; Napolitano et al., 2000).
Interestingly, G to T transversions induced by the same BaP adduct within
a TGT context (the adducted base shown in bold) were found to exclusively
require Pol V (Yin et al., 2004). The requirement of multiple polymerases
during lesion bypass, described as the DNA polymerase switch model
(Cordonnier and Fuchs, 1999; Woodgate, 1999), raises the question as to
the access of these polymerases to the different replication intermediates.
D. Regulating the Access of Pol IV to Replication Intermediates
1. Interaction between SOS Polymerases and Clamp is Essential for

Translesion Synthesis and Mutagenesis
As discussed in Section B.2, the Pol III holoenzyme processivity factor
(the clamp) interacts directly with Pol IV, largely increasing its
affinity for the substrates, synthesis efficiency, and apparent processivity
in vitro. A small peptide (LVLGL), at the extreme C terminus of Pol IV,
was shown to be essential for this interaction (Lenne-Samuel et al., 2002).
Because the deletion of this motif abolishes the interaction between Pol IV
and without affecting Pol IV’s intrinsic polymerase activity, it was possi-
ble to directly test the potential role of this interaction in vivo. The results
obtained clearly demonstrate that the C-terminal peptide is essential
for both B[a]P-guanine adduct bypass and the spontaneous mutator
phenotype of Pol IV in vivo (Lenne-Samuel et al., 2002). Concomitant to
these studies, Dalrymple et al. (2001) defined a consensus sequence,
QL(S/D)LF, for most of the known eubacterial -interacting proteins,
including E. coli Pol II and V. This allowed the construction of polB
and umuC alleles that are mutated in the clamp interacting motifs.
These mutants led to the demonstration that, similar to Pol IV, Pols II
and V lesion bypass activities require a functional interaction with the
clamp (Becherel et al., 2002). Similar interactions between the eukaryotic
PCNA processivity clamp and eukaryotic Y polymerases , , and have also
been demonstrated (see chapters by Vaisman, Lehmann and Woodgate
and Ohashi and Ohmori). From these studies it can be inferred that, in vivo,
the processivity clamps play a central role in lesion bypass.
2. Structural and Biochemical Studies Suggest Competition between

Polymerases for Binding to the clamp
All five DNA polymerases of E. coli interact with the clamp (Bonner
et al., 1992; Lopez de Saro and O’Donnell, 2001; Stukenberg et al., 1991;
Tang et al., 2000; Wagner et al., 2000). With the exception of Pol I, all these
interactions involve an identified motif that resembles the consensus

sequence defined by Dalrymple et al. (2001), thereby indicating competi-
tion of these polymerases for binding to the clamp. In an effort to gain
details on the interaction between Pol IV and , Burnouf et al. (2004)
solved the crystal structure of a complex between the clamp and the 16
residues C-terminal peptide (P16) of Pol IV, which contains the five last
amino acids essential for the interaction (Lenne-Samuel et al., 2002). The
authors showed that the seven C-terminal residues bind to a hydrophobic
pocket located at the surface of the molecule between subdomains II
and III (Fig. 5).
Interestingly, this region strictly corresponds to the previously identi-
fied binding site of another ligand, the subunit of the complex
( Jeruzalmi et al., 2001). The subunit binds to via the previously
mentioned consensus sequence (QAMSLF), and both peptides adopt a
very similar conformation (Fig. 5). Biochemical studies further showed
that all five E. coli DNA polymerases (as well as the subunit) bind to the
same site on (Burnouf et al., 2004; Lopez de Saro et al., 2003). Particu-
larly, the Pol IV P16 peptide is able to completely inhibit stimulation by
the clamp of Pol III subunit DNA synthesis in vitro (Fig. 6; Burnouf
et al., 2004). Similarly, a peptide derived from the binding site of Pol III
subunit to the clamp inhibits the loading of the clamp by the
complex as well as the -dependent Pol I DNA synthesis (Lopez de Saro
et al., 2003).
The fact that the same binding site on the clamp mediated the
interaction with many enzymes involved in DNA metabolism, including
essential ones such as the Pol III and subunits, leads the authors of
these studies to conclude that this hydrophobic pocket may represent an
attractive target for the development of a novel class of therapeutic agents
(Burnouf et al., 2004; Lenne-Samuel et al., 2002; Lopez de Saro et al., 2003).
Similarly, a ‘‘common’’ interacting pocket is conserved in PCNA, the
eukaryotic counterpart of the clamp, and may be used as a target for
the design of DNA replication inhibitors (Burnouf et al., 2004). These
studies also point to the fact that regulating access of the different
specialized polymerases to a replication fork may be simply mediated by
the relative affinities of the different partners for the processivity clamp.
Additional partners, such as RecA protein in E. coli, may also selectively
modulate the access of specialized polymerases to bypass intermediates
(see Section II). Moreover, posttranslational modifications of eukaryotic
PCNA (Haracska et al., 2004; Hoege et al., 2002; Kannouche et al., 2004;
Plosky and Woodgate, 2004; Stelter and Ulrich, 2003) or the existence of
alternative clamps such as the Rad9—Rad1-Hus1 complex in eukaryotes
(Kai and Wang, 2003, and references herein) and * in prokaryotes
244 FUCHS ET AL.
FIG. 5. Structure of Dpo4 polymerase and of the Pol IV and subunit -binding
peptides bound to the clamp. (A) Crystal structure of Dpo4. The DNA and nucleotide
in the ternary complex with Dpo4 are removed for clarity (Ling et al., 2001). The four
structural domains common among Y polymerase are shown in red (palm), blue
(Paz-Elizur et al., 1996; Skaliter et al., 1996a,b) potentially offer additional

ways to differentially recruit specialized DNA polymerases.
The multimeric nature of processivity clamps (review by Bruck and
O’Donnell, 2001) such as the subunit in E. coli (homo-dimer) and PCNA
in eukaryotes (homo-trimer) directly led to the formal possibility that two
or three potentially distinct, interacting proteins may bind simultaneously
to the clamp or PCNA, respectively thus constituting a ‘‘tool-belt’’
(Becherel et al., 2002). Although this possibility remains to be demon-
strated, some studies support this notion. First, as mentioned above,
homo-trimeric * clamps, which arise from the translation of the C-
terminal 2/3 of the dnaN gene after the induction of the SOS response
(Paz-Elizur et al., 1996; Skaliter et al., 1996a,b), may offer an additional
binding site to -interacting proteins such as MutS, Lig1, and the SOS
polymerases.
Second, Bunting et al. (2003) recently solved the crystal structure of a
complex between the E. coli clamp and the C-terminal domain of Pol IV,
comprising the so called ‘‘little finger domain’’ (LF domain; Ling et al.,
2001). This study shows that, in addition to the essential interaction
between the C-terminal Pol IV peptide and the hydrophobic binding
pocket on mentioned above, Pol IV makes an additional contact with
the clamp through an external face of the LF domain. Interestingly,
superimposition of the LF domain from a previously determined structure
of Dpo4 polymerase onto the ‘‘Pol IV-LF/’’ complex reveals that Pol IV
does not interact with the DNA primer template (‘‘OFF’’ position, Fig. 7;
Bunting et al., 2003). To gain access to the DNA, the additional protein–
protein interface between the LF domain and must be disrupted while
the interaction via the C-terminal peptide is maintained (‘‘ON’’ position,
Fig. 7).
(finger), green (thumb), and purple (little finger). The five conserved sequence motifs
shared by the Y-family polymerases are located in the palm, finger, and thumb
subdomains, which form the catalytic core domain. The peptide at the C terminus that
binds to the clamp is disordered in the crystal structure of Dpo4-DNA complex and
is therefore represented by the yellow dashed line. This figure is generated using
RIBBONS (Carson, 1987). (B) ribbon representation of the -ring with one P16 peptide
(the 16 C-terminal residues of Pol IV) bound at the interface of subdomains II and III of
monomer B. Only the seven C-terminal residues of the peptide were structured and
modeled in the density map (colored in yellow). (C) detailed stereo view of peptide P16/
monomer interaction and comparison with the subunit. The accessible surface of the
atoms in the -binding pocket is shown with hydrophobic residues in white, oxygen
atoms in red, nitrogen atoms in blue, and sulphur atoms in orange. P16 peptide is shown
in yellow, and the -interacting peptide of subunit is in blue. Part B was made with
Pymol (DeLano, 2002). Residues 10–16 from P16 correspond to residues 345–351 of full-
length Pol IV. (Adapted from Burnouf et al., 2004, with permission.) (See Color Insert.)
246 FUCHS ET AL.
FIG. 6. Inhibition of the -dependent activity of Pol III subunit by the Pol IV -
binding peptide (P16). A SSB-coated synthetic 32P-labeled primer/template duplex
(1 nM) allowing stable loading of the clamp is preincubated with (5 nM as a dimer;
lanes 5–8 and 13–16) or without (lanes 1–4 and 9–12) the processivity factor, the
clamp loader (1 nM), and increasing amounts (0, 1, 10, and 25 M final concentra-
tions) of either control peptide (that does not contain the -binding motif; lanes 1–8)
or P16 (the 16 C-terminal residues of Pol IV, which include the -binding motif; lanes
9–16). The DNA synthesis activity of the Pol III subunit on these substrates is then
assayed in the presence of all four dNTPS (200 M) for 1 minute at room temperature.
As shown in the left panel, the -independent activity of Pol III subunit, characterized
by the appearance of elongation products of no more than 12 nucleotides, is not
affected by the presence of either control of P16 peptides (lanes 1–4 and 9–12). In
contrast, although the -dependent activity of the subunit (characterized by
elongation products longer than 12 nucleotides) is not altered by the presence of
the control peptide (lanes 5–8), increasing amounts of P16 peptide lead to almost
complete inhibition of this specific activity (lanes 13–16). A quantitative analysis of the
experiment is shown in the right part of the figure (black and open squares represent
the calculated ratio of -dependent to -independent Pol III subunit activity in the
presence of indicated concentrations of control and P16 peptides, respectively).
(Adapted from Burnouf et al., 2004, with permission.)
FIG. 7. ‘‘ON-OFF’’ model for Pol IV bound to the clamp. Model of a Pol IV type-Y
DNA polymerase (red) bound to the clamp (gold) in the inactive, ‘‘locked-down’’
position (‘‘OFF’’ position, left part of the figure). The position of the polymerase was
modeled by superimposing the little finger (LF) domain of the archaeal Dpo4 enzyme
from the DNA complex (PDB code: 1JXL) onto the Pol IV-LF (blue) in the complex
with the clamp described in Bunting et al. (2003). Modeled in this position, the
polymerase makes no steric clashes with the clamp but cannot access the primer–
template junction (primer strand in purple, template strand in green). As far as the
clamp surface is not obstructed, such a complex may accommodate a second
polymerase bound to the second monomer of . Transition to a productive complex
(‘‘ON’’ position, right part of the figure) necessitates the disruption of the substantial
protein–protein interface between the clamp and the LF domain of the Pol IV
polymerase. In this ‘‘ON’’ configuration, contact with the clamp is maintained by the
C-terminal clamp-binding peptide (blue), which tethers the enzyme to the replication
complex. (Bunting, K. A., Roe, S. M., and Pearl, L. H. (EMBO) 20, 5883–5892 (2003);
advance online publication, [doi:10.1038/nature xxxxx]) (See Color Insert.)
The ‘‘OFF’’ position may also be seen as a way to bind more than one
ligand on a single multimeric clamp, with the polymerase being main-
tained away from the primer terminus while being present at a high local
concentration. Finally, the occurrence of such a ‘‘tool belt’’ structure is
strongly supported by the recent findings of Dionne et al. (2003), who
showed that the heterotrimeric equivalent of PCNA in the archeon S.
solfataricus is able to accommodate the simultaneous binding of three
distinct partners, DNA polymerase B1, ligase I, and Fen I. Such a multi-
functional complex ‘‘would facilitate a tight coupling of DNA synthesis
and Okazaki fragment processing’’ in this archeon (Dionne et al., 2003).
248 FUCHS ET AL.
II. DNA POLYMERASE V, THE UMUDC GENE PRODUCT
A. Genetic Requirements of Induced Mutagenesis

Chemical and UV irradiation physically alter DNA bases that can
be converted into mutations on replication. Although many ‘‘bulky’’
lesions block DNA replication, some base modifications may simply
change the coding properties of bases without blocking replication, thus
inducing mutations during normal replication without any additional pro-
teins (direct-acting mutagens such as alkylating agents producing O6-
Me-guanine or 04-Me-thymine adducts). In addition to these ‘‘small’’
alkylation lesions, replicative DNA polymerases may perform bypass of
‘‘bulky’’ adducts in vivo on their own. For instance, the adduct formed by
the chemical carcinogen N-2-aminofluorene bound to the C8 position of
guanine (G-AF), despite its bulkiness, is easily bypassed by the replicative
polymerases (Bichara and Fuchs, 1985; Koffel-Schwartz et al., 1996). In
contrast, the closely related G-AAF adducts, which contains an additional
N-acetyl group, is a strong replication block that cannot be bypassed by
replicative polymerases (Belguise-Valladier et al., 1994; Lindsley and Fuchs,
1994). For all lesions that block replication (indirect-acting mutagens such
as UV-light induced lesions, polycyclic hydrocarbon adducts, etc.), genetic
studies have revealed that mutagenesis requires additional gene products;
namely, those encoded by the umuDC and recA loci in E. coli (Blanco et al.,
1982; Dutreix et al., 1989; Kato and Shinoura, 1977; Steinborn, 1978; Sweasy
et al., 1990). Although most studies were initially based on UV light as the
mutagenic agent, many chemicals were subsequently shown to exhibit
similar genetic requirements. As far as replication-blocking agents are
concerned, RecA protein is the central component of the cellular response
to this class of DNA-damaging agents (for a recent review, see Courcelle and
Hanawalt, 2003). The key molecular intermediate is RecA*, the so-called
activated form of RecA, which is produced when RecA monomers polymer-
ize along single-stranded DNA, forming a nucleoprotein filament. Muta-
genesis was found to require both UmuC and UmuD0 , a proteolytic
fragment of UmuD (Burckhardt et al., 1988; Nohmi et al., 1988; Shinagawa
et al., 1988). The processing of UmuD into UmuD0 requires the interaction
of UmuD2 with the RecA nucleoprotein filament. This interaction stimu-
lates a latent ability of UmuD2 to autodigest, resulting in the removal of its
N-terminal 24 amino acids. In addition to derepression of the SOS response
by mediating cleavage of the LexA repressor and activating UmuD0 by
mediating cleavage of UmuD, it is believed that RecA protein has an
additional and direct role in promoting mutagenesis (Dutreix et al., 1989;
Sweasy et al., 1990). More recently, the clamp, the replication processivity
factor, was shown to be an essential cofactor for umuDC-mediated mutagen-

esis (Becherel et al., 2002). On the basis of a wealth of genetic data, it was
generally assumed that the role in mutagenesis of the umuDC gene products
was to transiently alter the properties of the replication complex as to allow
the replication machinery to proceed through damaged template bases.
For many years, the major barrier in studying umuDC-dependent TLS
in vitro was the difficulty in obtaining soluble UmuC protein. UmuC was
first purified from insoluble inclusion bodies in the laboratory of the late
Harison Echols by using a denaturation–renaturation protocol (Woodgate
et al., 1989). Using renatured UmuC, it was demonstrated that monomeric
UmuC interacts with a homodimer of UmuD0 to form a heterotrimeric
UmuD0 2C complex (Woodgate et al., 1989). Replication experiments
with UmuD0 2C demonstrated that the Umu complex, along with RecA
and PolIII, could bypass a synthetic abasic site in vitro (Rajagopalan et al.,
1992). A native UmuD0 2C complex was first purified by expressing recom-
binant UmuD0 and UmuC (Bruck et al., 1996) in a strain carrying chromo-
somal deletion of the umu locus (Woodgate, 1992). Analysis of the
biochemical properties of the native UmuD0 2C complex allowed Tang
et al. (1998) to recapitulate the earlier in vitro studies with denatured/
renatured UmuC from the Echols lab, but they also raised the intruiging
possibility that the Umu complex may possess intrinsic polymerase activity
(Tang et al., 1998).
However, at that time, it was impossible to rule out the possibility that
the polymerase activity was not from a minor contaminant of Pol II or Pol
III. Indeed, using a different approach in which soluble UmuC was
expressed as a Maltose binding protein (MBP) fusion, Reuven et al.,
(1998) did not detect any intrinsic polymerase activity associated with
the MBP-UmuC fusion (Reuven et al. 1998). However, the following year
Tang et al., purified native UmuD0 2C from strains lacking Pol II and
carrying a thermosensitive Pol III and clearly demonstrated that UmuD0 2
possessed intrinsic polymerase activity and, as a consequence, was called
Pol V (Tang et al., 1999). Similarly, the MBP-UmuC fusion protein was also
found to possess intrinsic polymerase activity (Reuven et al., 1999).
With the discovery of DNA polymerases specialized in TLS, it became
clear that lesion bypass and mutagenesis would entail several DNA poly-
merase switches, with the replicative DNA polymerase being transiently
replaced in the vicinity of the lesion by one (or several) specialized
polymerases before resuming high-fidelity replication. This new paradigm
for the mechanism of TLS and mutagenesis was dubbed the ‘‘DNA
polymerase switch model’’ (Cordonnier and Fuchs, 1999).
Next, we summarize the properties of Pol V and discuss some of the
conflicting results that have been reported over the last several years with
250 FUCHS ET AL.
respect to Pol V biochemistry. We will describe a ‘‘minimal’’ lesion bypass

assay that involves a long, circular, single-stranded template onto which
the essential cofactors for Pol V-mediated TLS, that is, the clamp and
RecA, are stably assembled. In a second part, we will summarize a recent
attempt to reconstitute the whole process of TLS in vitro, recapitulating
the switches between the replicative Pol III holoenzyme and the poly-
merases specialized in lesion bypass. Other aspects of umuDC biology,
namely, its regulation via proteolysis and its activity in a checkpoint-type
response, will not be discussed in this chapter (see the following recent
papers for these aspects: Gonzalez and Woodgate, 2002; Opperman et al.,
1999; Sutton et al., 1999).
B. Biochemical Properties of Pol V

In vitro, Pol V was shown to be able to bypass a TT cis-syn cyclobu-
tane dimer and TT (6-4) photoproduct with insertion specificities similar
to those observed in vivo, thus establishing the physiological significance
of Pol V (Tang et al., 2000). As already discussed in the literature (Sutton
et al., 2000; Walker, 1998), because of major differences in their experi-
mental design, the groups of Goodman and Livneh reported different
biochemical requirements for Pol V–mediated in vitro bypass. We have
recently reinvestigated the biochemical properties of native Pol V (Fujii
et al., 2004). Our results shed some light on the previously published
discrepancies, as we will outline below.
In addition to obvious differences in their respective Pol V preparations as
mentioned above, the groups of Livneh and Goodman used different DNA
substrates (Fig. 8). Livneh and colleagues used a gapped circular plasmid
with a single lesion located in a 350-nucleotide-long, single-stranded DNA
gap (Tomer and Livneh, 1999). In contrast, Goodman and colleagues used
primed linear templates, either a 240-mer synthetic oligonucleotide or a 7.2-
kb linear single-stranded phage DNA. In both cases, the single lesion was
located only about 50 nucleotides from the 50 -end of the single-stranded
template (Tang et al., 1999). Our own experimental design involves a
primed, 2.7-kb-long, single-stranded circular plasmid DNA (Napolitano
and Fuchs, 1997).
Several common replication-blocking lesions were used by the three
groups; namely, synthetic AP sites (Reuven et al., 1999; Tang et al., 1999),
TT cis-syn cyclobutane dimer (Fujii et al., 2004; Tang et al., 2000), TT (6-4)
photoproduct (Fujii et al., 2004; Tang et al., 2000), and the covalent adduct
formed by the chemical carcinogen N-2-acetylaminofluorene with guanine
(G-AAF) (Fujii et al., 2004). Although native Pol V (Fujii et al., 2004; Tang
et al., 2000) and UmuC protein alone (Reuven et al., 1999) are able to copy
FIG. 8. Basic requirements for Pol V–mediated translesion synthesis in vitro: sorting
out the published literature. The groups of Dr. Goodman (Goodman, 2002), Dr.
Livneh (Livneh, 2001), and ourselves (Fujii et al., 2004) have published work on the
reconstitution of lesion bypass using Pol V and accessory factors. As outlined in this
figure, the three studies differ in (i) the nature of the Pol V preparation and (ii) the
structure of the primer template. The length of the single-stranded region downstream
from the lesion site that appears to be a critical parameter (see text) is indicated for
each substrate. Except for RecA protein that was found to be essential in all three
studies, differences with respect to the requirements of SSB protein, ATP or ATP- S,
and the clamp have been reported as discussed in the text and summarized in this
figure.
undamaged DNA without any additional cofactor, TLS requires additional

factors. All three groups found that RecA protein is essential for lesion
bypass; however, some discrepancies with respect to the nature of the
nucleotide triphosphate cofactor that is required to activate the RecA
filament were reported. With the natural ATP cofactor, Goodman and
Livneh groups reported low or high Pol V TLS activity, respectively (Pham
et al., 2001; Reuven et al., 2001). With the slowly hydrolysable ATP- S
analog, opposite results were reported, Livneh’s group reported severe
inhibition, whereas Goodman’s group found high Pol V activity (Pham
et al., 2001; Reuven et al., 2001). In our hands, both ATP and ATP- S were
found to support high Pol V–mediated TLS activity provided that SSB
protein was added when the RecA filament is activated with ATP-gammaS
(Fujii et al., 2004). We suggest that SSB is required to disrupt the
252 FUCHS ET AL.
‘‘stablilized RecA filament’’ (Goodman, 2002; Pham et al., 2001) that is

formed in the presence of ATP- S. In contrast, in the presence of the
physiologically relevant RecA/ATP filament, SSB is not required for Pol V
bypass activity (Fig. 9). This latter finding is in striking contrast with the
findings of both Livneh and Goodman groups, who reported SSB to be an
essential component of the Pol V–mediated lesion bypass reaction (Pham
et al., 2001; Reuven et al., 2001). Goodman and coworkers suggested that
SSB is necessary, acting together with the advancing Pol V molecule as a
‘‘locomotive cowcatcher’’ that dissociates RecA monomers from the 30 end
of the filament (Pham et al., 2001). In contrast, our data indicate that Pol V
can copy a single-stranded template covered with RecA in the absence of
SSB (Fujii et al., 2004). To maintain a permanent contact between Pol V
and the tip of the RecA filament, we suggest that the RecA filament ‘‘slides
back’’ in a 30 ! 50 direction, with the RecA monomer dissociating from the
50 -end of the filament in an ATP-catalyzed reaction (Fig. 10b).
In vivo, another cofactor essential for Pol V–mediated lesion bypass is
the clamp, the replication processivity factor (Becherel et al., 2002). The
clamp is an annular structure that encircles and freely slides along DNA
(Kong et al., 1992). Therefore, its stable loading is best achieved on a
circular DNA substrate that prevents it from sliding off the template. Using
such a circular template, we found that Pol V–mediated TLS is highly
stimulated (
100-fold) by the presence of the clamp (Fujii et al., 2004).
Initially, Livneh reported that Pol V–mediated TLS only required a basic
four-component reaction set, including UmuC, UmuD0 , RecA, and SSB
(Reuven et al., 1999). In a subsequent study, this group observed a three-
fold stimulation of TLS in the presence of the clamp (Maor-Shoshani
and Livneh, 2002). Goodman’s group initially observed that and highly
stimulate Pol V’s bypass activity (Tang et al., 1999), but that the stimula-
tion was partially abrogated by the use of a RecA filament stabilized with
ATP- S (Tang et al., 2000).
In conclusion, it appears that Pol V bypasses efficiently all lesions that
have been tested so far (AP sites, TT cyclobutane dimers, TT (6-4) photo-
products, G-AAF adducts), provided specific cofactors are present at the
lesion site. These cofactors are RecA and the clamp. Although it can be
speculated that the role of the clamp is to maintain Pol V in the vicinity
of the substrate to allow it synthesize a TLS patch that is sufficiently long to
allow subsequent elongation of the nascent primer by Pol III holoenzyme
(see next paragraph), the exact role of RecA still remains to be discovered.
One possibility may be that on interaction with the clamp, Pol V is
locked in a position that prevents it from contacting the primer-template,
an ‘‘OFF’’ position, as recently described for Pol IV (Fig. 7; Bunting et al.,
2003). The specific role of RecA may be to interact with Pol V, thus
FIG. 9. SSB protein is not required for robust Pol V-mediated TLS activity in the
presence of a RecA/ATP filament. In these experiments, the amount of RecA protein
(2 M) is stoichiometric with respect to the amount of single-stranded DNA present
in the reaction mixture (
5.4 M expressed in nucleotides), given that one RecA
protein covers
3 nucleotides. While TLS efficiently occurs in the absence of
SSB protein (34%), a low amount of SSB protein (10 nM corresponding, to less than
10% of the amount required for template saturation) stimulates the TLS reaction
(66%). In contrast, increasing amounts of SSB (300 nM) strongly inhibit lesion bypass.
The reaction conditions are as follows. A circular single-stranded template (
2.7 kb)
containing a single G-AAF adduct is primed with a 50 -32P-labeled 25-mer oligonucleo-
tide, the 30 extremity being located 8 nucleotides upstream from the lesion site (L-
8 primer). A typical reaction mixture contains the DNA substrate (2 nM) in 20 mM
Tris-HCl (pH 7.5), 4% glycerol, 8 mM DTT, 80 g/ml BSA, 8 mM MgCl2, dATP, dTTP,
dGTP, dCTP (each 0.1 mM) and ATP (2.5 mM). The DNA substrate is preincubated
with -clamp (50 nM as a dimer), -complex (10 nM), RecA protein, and SSB at the

indicated concentrations at 30 C for 10 minutes. Reactions were initiated by adding

Pol V (100 nM), and incubated for 20 minutes at 30 C. The reaction products were
digested by EcoR I and analyzed by electrophoresis on a 10% denaturing polyacrylamide
gel. The data are quantified as follows: primer utilization efficiency (initiation
percentage) is calculated as the ratio of all products above the primer divided by the
amount of total primer input. TLS efficiency (TLS%) is the ratio of all products
above L0 (excluded) divided by the extent of primer utilization (sum of all bands above
the primer band).
254 FUCHS ET AL.
FIG. 10. Switches between replicative and specialized DNA polymerases during
translesion synthesis. (a) Pol III holoenzyme is able to elongate a primer in the vicinity
of a lesion provided its 30 -extremity is located four or more nucleotides downstream
from the lesion site. If the primer is shorter, the proofreading exonuclease that is
associated with Pol III degrades the primer (Fujii and Fuchs, 2004). (b) Minimal
conditions for robust Pol V-mediated TLS. Two cofactors are essential for efficient Pol
V-mediated lesion bypass: (i) a DNA substrate onto which the clamp is stably loaded,
correctly positioning the polymerase to engage the nascent primer termi-

nus (‘‘ON’’ position). Although a long RecA filament activated with ATP is
most likely to be the physiologically relevant cofactor, it has recently been
suggested that mechanistically, a single RecA monomer may be sufficient
for Pol V–mediated bypass (Pham et al., 2002).
C. Switches Between Replicative and Specialized DNA Polymerases

During Lesion Bypass
In vivo, the process of lesion bypass involves the recruitment of one or
several specialized DNA polymerases that will temporarily replace the
replicative polymerase in the vicinity of the lesion site. Indeed, for most
replication-blocking lesions, the replicative polymerase will stop at the
position preceding the lesion in the template (position L-1). Although
for some lesions replicative polymerases may, in fact, be able to incorpo-
rate a nucleotide opposite the damaged template base, the kinetic barrier
for further elongation will trigger its excision by the associated proof-
reading function, thus leading to futile insertion/excision cycles. DNA
polymerases specialized in lesion bypass have the intrinsic capacity to copy
lesion-containing templates. In addition, as these polymerases lack an
associated proofreading exonuclease, they will not remove the newly
incorporated nucleotides. A key factor for successful translesion synthesis
will be to endowing these highly distributive enzymes with enough ‘‘pro-
cessivity’’ to add several nucleotides onto the primer at once. Let us define
and (ii) an extended single-stranded RecA/ATP filament assembled downstream from

the lesion site. For efficient bypass, Pol V needs to interact simultaneously with the
clamp and the 30 tip of the RecA filament. Formation of an extended RecA/ATP
filament and stable loading of the clamp are best achieved on long single-stranded
circular DNA templates. Under these conditions, SSB protein is not required for the
bypass reaction. We suggest that to maintain a permanent contact between the
advancing Pol V polymerase molecule and the 30 tip of the RecA filament, RecA
molecules dissociate from the 50 end of the filament. (c) Overall lesion bypass scenario
(Fujii and Fuchs, 2004): when Pol III associated with its clamp encounters,
a noncoding lesion in the template a RecA filament formed on the single-stranded
DNA region downstream the lesion site. This RecA filament, together with the clamp,
forms a structure-specific template to which the bypass polymerase Pol V binds and
mediates, in a single binding event—a TLS patch 20 nucleotides long on average (Fujii
and Fuchs, 2004). The size distribution of the patches generated by Pol V is such that
75% of the patches are longer than 5 nucleotides, thus allowing subsequent
elongation by the replicative polymerase. In contrast, the patches that are less than 5
nucleotides long (
25%) are degraded by the Pol III–associated proofreading function,
leading to an aborted bypass process.
256 FUCHS ET AL.
the ‘‘TLS patch’’ as being the fragment of DNA that is made by the
specialized DNA polymerase opposite a template lesion. The TLS patch
needs to be sufficiently long to prevent it from being degraded by the
proofreading function on rebinding of the replicative polymerase. As will
be discussed below, the interaction of the specialized DNA polymerases with
the general replication processivity factor, that is, the clamp, is essential for
this purpose. First, we determined the patch size made by Pol V, in a single-
binding event, under optimal conditions; that is, in the presence of an
activated RecA/ATP filament and the clamp (as in Fig. 9). For a single
G-AAF adduct, under standing-start conditions, we find that Pol V produces
a large distribution of TLS patches, ranging from 1 to 60 nucleotides long,
with the average length being about 20 nucleotides (Fujii and Fuchs, 2004).
Similar results were obtained with the TT cyclobutane and the TT (6-4)
photoproduct. In the absence of clamp, Pol V is completely distributive
and is therefore unable to participate in a successful TLS event (Fujii and
Fuchs, 2004). Second, we tested the capacity of Pol III holoenzyme (Pol III
HE), the replicative machinery in E. coli, to extend a primer when a lesion is
present in the template strand. It turns out that for a set of different lesions
(AP site, TT cyclobutane dimer, TT(6-4) photoproduct, and G-AAF adduct),
efficient primer elongation by Pol III HE requires the primer to reach 4–5
nucleotides beyond the lesion site (Fujii and Fuchs, 2004). On the basis of
structural studies, it was suggested that replicative-type DNA polymerases
possess a minor groove recognition domain that acts as a ‘‘sensor’’ capable
of discriminating between correctly and incorrectly paired nucleotides
within the four to five last base pairs (Kiefer et al., 1998). Disruption of these
minor-groove interactions caused by the presence of the lesion within the
four last base pairs of the nascent double-stranded DNA ( Johnson et al.,
2003) is likely to cause stalling of DNA synthesis and, as a consequence, to
activate degradation via proofreading. Under our experimental conditions,
it turns out that about 75% of the TLS patches made by Pol V when it
bypasses a single G-AAF adduct are longer than 5 nucleotides and will
therefore be converted into TLS events on rebinding of Pol III replicase
(Fujii and Fuchs, 2004). Conversely, about 25% of TLS patches are shorter
than 5 nucleotides and will be degraded by the proofreading function of Pol
III. These aborted TLS events will enter a new bypass attempt. The average
patch size of
20 nucleotides may appear as a reasonable trade-off between
ensuring efficient TLS (75%, in a single attempt) and preventing a heavy
load of untargeted mutations. The level of untargeted mutation made by Pol
V can be estimated as follows: Given its fidelity on undamaged DNA (103 to
104, Maor-Shoshani et al., 2000; Tang et al., 2000), Pol V will produce only
one untargeted mutation per 50–500 TLS events, a low level of point
mutations that will potentially be corrected by mismatch repair. We suggest
that the process of polymerase switching during TLS as described here in

E. coli may represent a simplified paradigm for lesion bypass in eukaryotes.
As already discussed here in section I.D.3, the control of polymerase
switching in yeast and human cells appears to be much more complicated,
as it involves PCNA and its posttranslationally modified forms as well as
alternative sliding clamps.
Acknowledgments
We gratefully thank Drs. Roger Woodgate and Wei Yang for critical reading and
suggestions.
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MAMMALIAN POL k: REGULATION OF ITS EXPRESSION
AND LESION SUBSTRATES
By HARUO OHMORI,* EIJI OHASHI,* AND TOMOO OGIÀ
*Institute For Virus Research,

Kyoto University, Sakyo-ku, Kyoto, 606-8507, Japan
À
Genome Damage and Stability Centre, University of Sussex,
Farmer, Brighton BN19RR, United Kingdom
I. Structures of the Genes and Proteins. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 265

A. Regulation of Expression of the Human POLK Gene and
the Mouse Polk Gene . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 265
B. Splicing Variants and Enzyme Activities . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 269
C. Conservation of the C2HC Zinc Cluster Sequence . . . . . . . . . . . . . . . . . . . . . .. . . . . . 271
II. Enzymatic Properties of Pol. . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 271
A. Is Pol an Inserter or an Extender?. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 271
B. What are the Cognate DNA Lesions for Pol? . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 273
III. Possible Mechanisms of TLS by Pol In Vivo . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 274
References . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 275
I. STRUCTURES OF THE GENES AND PROTEINS
A. Regulation of Expression of the Human POLK Gene and the Mouse

Polk Gene
Chromosomal DNAs in living organisms are continually exposed to a
vast variety of genotoxic agents from exogenous and endogenous sources.
Some environmental compounds such as polycyclic aromatic hydrocar-
bons (PAHs) are activated as mutagens in mammalian cells (Friedberg
et al., 1995). Among PAHs, benzo[a]pyrene (B[a]P) has been most exten-
sively studied because it is believed to be a potent carcinogen, especially
responsible for the p53 mutations detected in the lung tumors of smokers
(Denissenko et al., 1996). PAHs are activated by intracellular processes that
are mediated by the arylhydrocarbon receptor (AhR) (for a recent review,
see Nebert et al., 2004). AhR is called also as a dioxin receptor, because
dioxin has a much higher affinity to AhR. When a PAH compound enters
mammalian cells, it binds to AhR localized in the cytoplasm. The ligand-
activated AhR then moves to the nucleus, where the protein forms a complex
with Arnt (AhR nuclear translocator). The AhR-Arnt complex binds to
specific DNA sequences called XREs (xenobiotic responsive elements),
which are present in the promoter regions of PAH-inducible genes includ-
ing the mouse Cyp1a1 gene. The product of the Cyp1a1 gene is a cytochrome

266 OHMORI ET AL.
P450 protein (1A1) that metabolizes B[a]P and other PAHs to phenols
and dihydrodiols to excrete such lipophilic compounds from the inside of
cells. However, some of the metabolites become activated so as to attack
DNA to form a covalent linkage. B[a]P diol epoxide (BPDE, see Fig. 1),
the so-called ultimate carcinogen derived from B[a]P, introduces bulky
adducts predominantly at the N 2 position of guanine and less frequently
at the N 6 position of adenine.
Analysis of the mouse Polk genomic sequence revealed that the promoter
region contains two copies of XRE-like sequence (Ogi et al., 2001). In vitro
experiment with partially purified AhR and Arnt proteins showed that the
AhR–Arnt complex did bind to each of the two XRE-like sequences found in
the Polk promoter region, although less efficiently compared with the XRE
sequence in the Cyp1a1 promoter region. Furthermore, expression of
the mouse Polk gene was stimulated when wild-type mice were treated
with 3-methylcholanthrene (3MC), a PAH compound similar to B[a]P
(see Fig. 1). In contrast, such stimulation by 3MC was not observed in
AhR-knockout mice, whereas a basal level of the Polk expression was still
observed. AhR-knockout mice show no detectable Cyp1a1 expression with or
without 3MC treatment, and they are refractory to B[a]P-induced skin
tumors (Shimizu et al., 2000). The human POLK gene also has similar
XRE-like sequences in the promoter region. O-Wang et al. (2001) found
FIG. 1. Structures of benzo[a]pyrene, 3-methylcholanthrene and dioxin. Benzo[a]-

pyrene and 3-methylcholanthrene are PAH compounds, but dioxin is not. Benzo[a]pyrene
is converted to the ultimate carcinogen BPDE by Cyp1A1.
MAMMALIAN POL 267
that Pol was frequently overexpressed in human lung cancer tissues as

compared with a matched nontumor tissue counterpart. More recently, a
close correlation between elevated Pol expression and p53 inactivation in
human lung cancer tissues was found (Wang et al., 2004). In contrast
Velasco-Miguel et al. (2003) reported that the mouse Polk gene transcription
was regulated by the p53 gene both constitutively and following exposure to
selected DNA-damaging agents such as ultraviolet (UV) irradiation and
doxorubicin, whereas the human POLK gene expression was not induced
by such agents. The detailed mechanisms by which the mouse Polk and the
human POLK genes are regulated by p53 in different ways remain unclear,
because no sequence similar to the consensus p53 binding sequence has
been identified near their promoter regions.
Further analyses of the mouse Polk and human POLK genomic se-
quences revealed that another gene (COL4A3BP in humans and Col4a3bp
in mice) transcribed into the opposite direction is present in the immedi-
ate upstream of the genes. The initiation sites for the two divergent
transcriptions in the human and mouse genomes are separated by only
about 300 nucleotides, indicating that those genes share some regulatory
elements to bind common transcription factors. The human COL4A3BP
gene was initially identified as the GPBP (Goodpasture antigen binding
protein) gene, because the product was thought to bind to the C-terminal
region of the collagen IV alpha-3 chain, a Goodpasture antigen (Raya et al.,
1999). However, a recent paper demonstrated that the COL4A3BP gene
codes for CERT, a protein that mediates intracellular trafficking of the
lipid precursor ceramide (Hanada et al., 2003). At present, it is unknown
how the gene product carries out two such completely different functions.
Moreover, almost nothing is known about how the GPBP/CERT gene
expression is regulated, although the gene might be also under the
control of AhR–Arnt. It should be noted that many eukaryotic genes,
including those coding for DNA polymerase, have a gene in the immediate
upstream that is transcribed into the opposite direction (see Adachi and
Lieber, 2002). Another typical example for such a bidirectional gene
organization related to a lesion-bypass enzyme is the human XPV and
XPO5 genes, which code for Pol and Exportin 5, respectively.
Both of the human POLK and mouse Polk genes are ubiquitously
expressed with the highest expression in testis. Such expression patterns
are observed with many other genes involved in DNA repair and recombi-
nation functions. However, one unique feature of the human and mouse
genes encoding Pol is that the major transcripts found in the testes are
different in size from those found in other organs; the mRNAs abundantly
present in the testes are about 2.8 kb whereas those ubiquitously
expressed in many organs (including testis) are 4.2 kb (Gerlach et al.,
268 OHMORI ET AL.
1999; Ogi et al., 1999). The major difference between the two distinct
species of mRNAs resides in the 30 -untranslated region (30 -UTR).
Although the shorter 2.8-kb transcripts have a polyA stretch in the imme-
diate downstream of the translation stop codon, the longer 4.2-kb tran-
scripts have a long (1.4 kb) 30 -UTR containing multiple copies of AUUUA
sequence (Gerlach et al., 1999, see Fig. 2). The AUUUA sequence is an
essential and minimal unit of AREs (A+U-rich elements) that are frequent-
ly found in the 30 -UTRs of highly labile mRNAs, such as those encoding
cytokines, growth factors, and proto-oncogenes (Xu et al., 1997). ARE when
present in 30 -UTR is considered to make mRNAs unstable. The alternative
selection of the polyA site in the testes may render the 2.8-kb transcripts
FIG. 2. The genomic structure of the mouse Polk gene and splicing variants.
Transcription of the mouse Polk gene starts at two different sites, between which P1a is
ubiquitously used and P1b is almost exclusively in testis. Major transcripts found in testis
are 2.8 kb in length. Most of them have a polyA tail immediately after the translation
stop codon (1 and 2), in which the AAUGAA containing the stop codon should
correspond to the polyA signal sequence. Some transcripts lacked the translation stop
codon (as in variant 4), where the upstream AAUAAA sequence was used as the polyA
signal sequence. About 50% of transcripts in testis lacked the exon 7 (as in variant 3).
Longer transcripts of 4.2 kb in length were ubiquitously expressed, which included a
long 30 -UTR with multiple copies of ARE sequence. The coding region starts within
exon 2 and terminates within exon 15.
MAMMALIAN POL 269
lacking an ARE sequence more stable than the 4.2-kb transcripts and may
consequently result in the abundant accumulation of the 2.8-kb transcripts
in the testes. Many tissues may express only labile mRNAs to keep the
amount of Pol at a low level and avoid gratuitous mutations due to the
presence of an excess amount of the error-prone enzyme.
B. Splicing Variants and Enzyme Activities

Testis-specific regulation of the mouse Polk transcription is also observed
as splicing variants. RT-PCR experiments indicated that about 50% of the
human POLK-mRNAs from testis lacked the exon 7 of 240 nucleotides in
length (our unpublished results; see Fig. 2), and a minor proportion
of them lacked the exon 13 (Gerlach et al., 1999). The full-length forms
of the human and mouse Pol proteins consisted of 870 and 852 amino
acids, respectively (Figs. 2 and 3). When the mouse testis extracts were
reacted with anti-Pol antibody, which was raised against the peptide
antigen corresponding to the human Pol 558–571 sequence (identical
to the mouse 557–570 sequence), two different protein bands of 100 and
80 kDa were observed. The 100-kDa band probably corresponds to the
intact form of the mouse Pol protein, which is observed as the sole
product in other organs. The shorter, 80-kDa band was observed exclu-
sively in testis, which seems to be too small to be the product originated
from skipping of the exon 7. It is also unlikely that the 80-kDa band is
derived from the splicing variant lacking the exon 13, because the antigen
sequence taken to raise the antibody used in the experiment is encoded by
the exon 13.
The N-terminal half of Pol forms a catalytic core domain containing
multiple motifs that are shared among the Y-family DNA polymerases in-
cluding the Escherichia coli DinB (Pol IV) (Fig. 3). The C-terminal half is not
essential for the DNA polymerase activity per se, at least in vitro, but the region
would be necessary for the in vivo function because putative nuclear locali-
zation signal (NLS) and PCNA binding site (PBS) are found at the extreme C
terminus. Two copies of C2HC zinc cluster sequence, which is similar to that
found in RAD18 homologues, are found between the catalytic domain and
the C terminus. A truncated form containing the 1–560 residues, which was
overproduced by a baculovirus system, showed a DNA polymerase activity
in vitro that bypasses certain DNA lesions, as does the full-length protein
(Ohashi et al., 2000a,b), but a shorter form containing the 1-510 residues did
not show a DNA polymerase activity (Gerlach et al., 2001). Skipping exon 13
in the human POLK-mRNA should result in the deletion of the 511–830
residues, and therefore, the protein derived from the mouse splicing variant
lacking exon 13 is expected to be inactive as a DNA polymerase. Recently, we
270 OHMORI ET AL.
FIG. 3. The genomic structure of the human POLK gene and structure of the Pol
protein. The human POLK gene has a genomic structure very similar to that of the
mouse Polk gene. The N-terminal half of Pol contains multiple motifs (I–V) conserved
among Y-family DNA polymerases, which are required for the enzyme activity. NLS and
PBS at the C terminus represent putative nuclear localization signal and PCNA binding
sites. Splicing variants lacking exons 7 and 13 lack a part of the motifIV and two copies
of C2HC sequences, respectively. Various forms of Pol protein lacking the C-terminal
half were overproduced in Escherichia coli and assayed for DNA polymerase activity.
developed a system to overproduce the human Pol protein in E. coli cells.

The 1–560 protein overproduced in E. coli cells retained the DNA polymer-
ase activity, but deletion of the 232–311 residues corresponding to the
region encoded by exon 7 of the mouse Polk and human POLK genes
abolished the activity. Thus, neither of the two splicing variants observed
in mouse testis appears to direct a protein active as a DNA polymerase.
Mammalian Pol proteins have an N-terminal extension of about 100
residues that is not conserved among the Y-family proteins. We overpro-
duced the protein containing the 91–560 or 117–560 residues of human
Pol in E. coli. When measuring DNA polymerase activity in vitro, the 91–560
protein retained the enzyme activity, although weaker than the 1–560
protein, but the 117–560 protein did not show any activity (see Fig. 3).
MAMMALIAN POL 271
C. Conservation of the C2HC Zinc Cluster Sequence

Recently, Pol homologs in chicken and rat were identified (Okada et al.,
2002; the XP_342179 entry in the protein database). The amino acid
sequences of the C-terminal half domain in such homologs are variable,
except for the two C2HC-cluster, putative NLS and PCNA binding se-
quences. A DinB/Pol homolog is present in Schizosaccharomyces pombe
and Caenorhabditis elegans, but not in Saccharomyces cerevisiae or Drosophila
melanogaster. The hypothetical Pol homologs of S. pombe and C. elegans
contain the multiple motifs shared among the Y-family DNA polymerases,
but their protein sequences deduced by conceptual translation (e.g.,
P34409 and T41397, respectively, in the Swiss-protein database) do not
have a C2HC zinc cluster sequence in the C-terminal regions. However, the
absence of a C2HC motif in the S. pombe and C. elegans Pol homologs seems
to be a result of an error in the prediction of protein coding region by
computer programs, which neglected the presence of one additional exon
at the 30 -terminus in each case. The S. pombe Pol homolog actually contains
a total of 547 residues with a C2HC sequence (492-CPVC-X13-HVDLC-513)
(our unpublished results), instead of 493 residues, as described in the
T41397 entry (see Johnson et al., 1999). Similarly, the hypothetical C. elegans
Pol homolog (F22B7.6) should contain 596 residues with a C2HC motif
(519-CPIC-X13-HVDEC-540), instead of 518 residues described in the
P34409 entry. In fact, a hypothetical Pol homolog (CAE75018) of the
closely related nematoda C. briggsae contains 612 residues with a C2HC motif
(515-CPIC-X17-HVDEC-540). Thus, eukaryotic Pol homologs appear to
share a common basic architecture, including a catalytic domain, C2HC
(one or two copies), NLS, and PCNA binding site from the N to C terminus
(Fig. 3). The function of the C2HC motif remains to be determined.
II. ENZYMATIC PROPERTIES OF POL
A. Is Pol an Inserter or an Extender?

Thus far, at least four different groups have reported their experimental
results on in vitro TLS by Pol, all of which are consistent with stating that
the enzyme cannot bypass T-T cyclobutane pyrimidine dimer (CPD) in vitro
by itself (Gerlach et al., 2001; Johnson et al., 2000a; Ohashi et al., 2000a;
Zhang et al., 2000a). However, Pol-defective mutants derived from mouse
embryonic stem (ES) and fibroblast (MEF) cells or from chicken DT40
cells are moderately UV sensitive (Ogi et al., 2002; Okada et al., 2002;
Schenten et al., 2002), indicating that Pol is somehow involved in the
response to ultraviolet (UV)-induced DNA damages. Washington et al.
272 OHMORI ET AL.
FIG. 4. Structures of BPDE adducts and estrogen-derived adducts. BPDE forms

covalent linkage at the N 2 of guanine (major products) and N 6 of adenine (minor
products). Estrogen also forms similar adducts.
(2002) showed that Pol efficiently extended G and A (but less efficiently C
and T) placed opposite the 30 T of a T-T CPD, whereas the enzyme did not
insert any nucleotide opposite the 30 T of the same lesion or extend any base
placed opposite the 30 T of a (6–4) T-T photoproduct. In addition, they
found that Pol efficiently extended from base mispairs on undamaged
DNA. Subsequently, Haracska et al. (2002a) found that Pol efficiently
extended various nucleotides incorporated opposite O6-methly guanine
and 8-oxoguaine by Pol . Thus, they concluded that Pol should play a
role as an extender in translesion synthesis. However, the efficiency of Pol
or any other TLS enzyme in inserting a correct or incorrect nucleotide
opposite a DNA lesion site strongly depends on the species of the lesion.
In fact, the authors also noticed that Pol inserted A in preference to other
bases opposite 8-oxoguaine at 37% efficiency (in terms of relative
ratio of kcat/Km), compared with nondamaged G, whereas it inserted
C opposite O6-methyl guanine at 1% efficiency, still higher by more than
MAMMALIAN POL 273
two orders of magnitude than for other bases. We believe that the bypassing
efficiency of each enzyme in comparison with nondamaged template should
greatly vary on the extent of a distortion in the DNA structure caused by
the respective DNA lesion, and that the more important datum is the relative
efficiency of each lesion by a given TLS enzyme in comparison with that
of other TLS and replicative DNA polymerases.
As the mouse Polk gene expression was induced by 3MC, an analog of
B[a]P, which is believed to be a major causative agent of human lung
cancers, whether or not Pol could bypass DNA adducts generated by
B[a]P was thus examined. The results obtained by two groups indeed
showed that human Pol could bypass different stereoisomers of dG-N2-
BPDE (the major products generated by BPDE, see Fig. 4) by inserting the
correct C opposite the bulky lesions in preference to other bases (Suzuki
et al., 2002; Zhang et al., 2000a, 2002). Furthermore, Pol bypassed dG-N2-
BPDE more efficiently than human Pol and Pol (alone or in combina-
tion with the yeast Pol ) (Rechkoblit et al., 2002). Very interestingly, Pol
inserted A more efficiently than C opposite dG-N2-BPDE (Chiapperino
et al., 2002; Rechkoblit et al., 2002; Zhang et al., 2000b). Pol inserted C
opposite dG-N2-BPDE adducts at 1% efficiency of nondamaged G, much
lower when compared with the fact that Pol can insert A opposite the 30 T
of a T-T CPD at the same efficiency as it does with nondamaged template
(Johnson et al., 2000b). Nevertheless, we believe that Pol plays a critical
role in the response to B[a]P–induced DNA damages, because mouse ES
cells with a Polk-defective mutation showed a hypersensitivity of B[a]P,
generating more mutations than the parental cells (Ogi et al., 2002).
Furthermore, the spectrum of B[a]P-induced mutations in Polk-defective
cells was different from that in the wild-type cells; G-to-T transversions
predominated (70%) among the mutations observed in Polk-defective
cells, whereas G-to-T and A-to-G substitutions occurred at an equal fre-
quency of 30% of total mutations in the parental cells. Such in vivo
results strongly indicated that Pol contributes to error-free bypass of dG-
N2-BPDE adducts and that in the absence of Pol, another enzyme,
probably Pol, inserts A opposite the adducts to generate G-to-T transver-
sions. This situation is reminiscent of that in the XPV cells lacking Pol,
where other TLS polymerases are involved in error-prone bypass of UV-
induced DNA damages.
B. What are the Cognate DNA Lesions for Pol?

The E. coli Pol IV (DinB) protein is also able to bypass dG-N2-BPDE
adduct by inserting the correct C opposite the lesion in vitro (Shen et al.,
2002). The ability to correctly bypass dG-N2-BPDE adduct is apparently
274 OHMORI ET AL.
conserved among the DinB/Pol homologs from E. coli to mammals. A

structure around the active sites conserved by the DinB/Pol homologs,
but not by Pol or Pol, probably enables the bulky dG-N2-BPDE adduct
to form a base paring with C. However, the ability of the E. coli Pol IV to
bypass such lesions should have little biological role because E. coli cells do
not have an enzyme to activate B[a]P into BPDE. Similarly, it seems
unlikely that mammalian Pol enzymes are conserved just for bypassing
B[a]P-induced DNA damages. Fischhaber et al. (2002) reported that
human Pol was able to insert A opposite thymine glycol (Tg) at 0.2%
5% efficiency of nondamaged thymine in vitro. This seems to be consis-
tent with the finding that the mouse Polk gene expression is induced
by UV radiation and doxorubicin, both of which are known to generate
Tg and other lesions in DNA (Velasco-Miguel et al., 2003). More recently,
Suzuki et al. (2004) found that human Pol could efficiently bypass
through N2-[3-methoxyestra-1,3,5(10)-trien-6-y1]-20 -deoxyguanosine (dG-
N2-3MeE) by inserting C opposite the lesion at 33% efficiency of nonda-
maged G. dG-N2-3MeE is a model compound used in place of N2-(2-
hydroestron-6-yl-)-20 -deoxyguanosine (2-OHE-N2-dG; see Fig. 4), a pro-
duct generated by the reaction between DNA and estrogen-2,3-quinone,
which is a metabolite generated by oxidation of estrogen. Because Pol is
highly expressed in the adrenal cortex of embryonic mice (Velasco-Miguel
et al., 2003) a site of active steroid biosynthesis, such estrogen-derived DNA
adducts, especially those formed at the N2 position of guanine with some
structural similarity to dG-N2-BPDE, are good candidates for endogenous
DNA damages that Pol could cope with. To conclude that thymine glycol
and estrogen-modified guanine are cognate lesions for Pol, in vivo
experiments demonstrating that Pol-deficient cells are defective for the
bypass of such adducts are required.
III. POSSIBLE MECHANISMS OF TLS BY POL IN VIVO

The most important question for the mechanism of TLS in vivo is how a
TLS enzyme replaces the replicative DNA polymerases stalled at a DNA
lesion site, performs the lesion-bypass reaction, and is then replaced by the
replicative enzyme again. One hot issue in this regard is the finding that
monoubiquitination of PCNA by the RAD6-RAD18 complex is required
for S. cerevisiae Pol to function in vivo at a lesion site (Hoege et al., 2002;
Steler and Ulrich, 2003; see the chapter by Xiao). Thus far, the interac-
tions between TLS enzymes and PCNA have been studied, using unmodi-
fied PCNA (Haracska et al., 2001a, b, c, and 2002b), so that they should be
reexamined with ubiquitinated PCNA. In contrast, a study on the S. pombe
DinB homolog suggested that its functioning needs the activity of the
MAMMALIAN POL 275
RAD9-RAD1-HUS1 (9-1-1) complex (a complex similar to PCNA) and the

RAD17-RFC(2-5) complex (the loader of the 9-1-1 complex to DNA) (Kai
and Wang, 2003). It is still an open question whether the human Pol
prefers ubiquitinated PCNA to the unmodified one or uses the 9-1-1
complex instead of unmodified and ubiquitinated PCNA.
Studies to search for Pol-interacting proteins revealed that Pol
interacts with a C-terminal region of REV1 (Guo et al., 2003; Ohashi et al.,
2004), which has been known to interact with REV7, the noncatalytic
subunit of another TLS enzyme Pol (Murakumo et al., 2001). Interestingly,
REV1 has a BRCT domain near the N terminus, which may function as an
interaction domain with another protein (for more details, see the chapter
by Lawrence). Because the C-terminal region of REV1 also interacts with
Pol and Pol, it seems likely that REV1 plays a key role, for example, as a
scaffold protein in a multiprotein complex for TLS in vivo.
We are still left with many unanswered questions regarding the in vivo
TLS mechanisms. For example, how is the RAD6-RAD18 complex
activated on DNA damaging? Is deubiquitination of PCNA required for
the replicative DNA polymerase to resume DNA synthesis when lesion-
bypass is completed? What is the mechanism for recruiting an appropriate
enzyme to a given species of DNA lesion. Obviously, further intensive
investigations are required to resolve these intriguing questions.
Acknowledgments
The studies in our laboratory cited here are supported in parts by Research-in-Aid Grants
(to H.O. and E.O.) from the Ministry of Education, Culture, Sports, and Science of Japan.
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DNA POSTREPLICATION REPAIR MODULATED BY
UBIQUITINATION AND SUMOYLATION
By LANDON PASTUSHOK AND WEI XIAO
Department of Microbiology and Immunology, University of Saskatchewan,

Saskatoon, Canada
I. Introduction . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 279
II. DNA Postreplication Repair in Prokaryotes . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 280
III. DNA Postreplication Repair in Eukaryotes. . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 281
IV. Ubiquitination . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 283
V. Protein Conjugation in PRR. . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 286
A. Rad6-Rad18. . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 286
B. Mms2-Ubc13-Rad5 . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 288
C. Ubc9-Siz1 . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 291
VI. Postreplication Repair via Covalent Modifications of PCNA . . . . . . . . . . . . . . . .. . . . . . 292
VII. Functional Conservation of Eukaryotic Postreplication Repair . . . . . . . . . . . . .. . . . . . 295
VIII. Future Directions. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 297
IX. Conclusions . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 300
References. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 301
I. Introduction
To ensure the viability of itself and its progeny, the living cell has
developed ways to reduce or avoid detrimental changes to its genetic
material. Because of the numerous external and internal agents that act
on and modify DNA, the incredible task of ensuring DNA fidelity and the
survival of individual organisms is made possible by a variety of DNA repair
and replication processes. In most cases, these processes are well under-
stood and, as such, are the major focus of other chapters in this book.
Conversely, DNA damage-tolerance pathways such as postreplication
repair (PRR) in eukaryotes are not yet well defined. Part of the difficulty
in this case is that PRR is not superficially considered an actual DNA repair
mechanism. PRR itself does not result in the physical removal of DNA
lesions but exists as a means to circumvent the severe consequences of
replication blocks that otherwise lead to cell death. This has made it
difficult to describe PRR in the context of a pathway or biochemical
activity. Instead, PRR loosely refers to events whereby damage-induced
single-stranded DNA gaps are somehow converted into double-stranded
DNA after replication. To advance our understanding of the genetics
and possible biochemical processes of PRR, the yeast Saccharomyces
cerevisiae has been the most studied eukaryotic model organism. Recent

280 PASTUSHOK AND XIAO
experimentation in the budding yeast has finally provided a functional

and mechanistic context for this often puzzling enigma. This chapter will
focus on yeast PRR from a protein-based perspective, with emphasis on the
covalent protein modification of proliferating cell nuclear antigen
(PCNA) by ubiquitin (Ub) and a small Ub-like modifier (SUMO). Readers
may wish to refer to recent reviews dealing with the genetic analysis of PRR
in particular (Broomfield et al., 2001) and the DNA damage tolerance
network in general (Barbour and Xiao, 2003).
II. DNA Postreplication Repair in Prokaryotes

It can perhaps be argued that the mechanism of DNA damage tolerance
has been best described in prokaryotes such as Escherichia coli. Indeed,
research into the SOS response and the RecA protein has led to the
discovery of physiological consequences comparable to PRR in S. cerevisiae.
However, although parallels can be drawn between recombination and
mutagenic translesion DNA synthesis (TLS) in each case, the regulatory
mechanisms and molecular architecture are very different. Consequently,
a brief overview of DNA damage tolerance in prokaryotes provides an
interesting perspective and raises interest in the remarkable molecular
evolution of PRR regulatory components in eukaryotes.
The crux of DNA damage tolerance in E. coli lies in the RecA protein,
which plays both a regulatory role in controlling the SOS response and a
physical role in repairing and bypassing damaged DNA. When the repli-
cation machinery encounters a block, such as a lesion generated by DNA
damage, synthesis is reinitiated downstream, and the result is a single-
stranded DNA (ssDNA) gap (Rupp and Howard-Flanders, 1968). In its
regulatory role, RecA acts as a sensor that recognizes these regions via
ssDNA binding affinity and consequently transforms itself to an activated
form, RecA* (Salles and Defais, 1984). RecA* acts as a coprotease that
stimulates LexA to autodigest (Little, 1984). Under noninduced condi-
tions, LexA forms a homodimer that binds to ‘‘SOS boxes’’ found in the
promoter regions of SOS regulon genes and represses, in varying degrees,
the expression of over 20 genes involved in many facets of DNA repair and
replication, including those for postreplication repair. Thus, when RecA*
stimulates the autodigestion of the LexA repressor, the SOS regulon genes
including lexA and recA are expressed at increased levels. As part of a
negative feedback loop, the newly synthesized LexA and RecA proteins
ensure a rapid return to the repressed state once the activating signal
(ssDNA gaps) is removed from the cell.
Similar to its role in controlling SOS response after DNA damage, a
second means of regulation by RecA in prokaryotic PRR also involves the
DNA POSTREPLICATION REPAIR 281
coprotease activity of RecA*. The umuC and umuD genes, also under
the control of SOS response, encode two subunits of a mutagenic transle-
sion polymerase, Pol V (Reuven et al., 1999; Tang et al., 1998, 1999). On
DNA-damage treatment, RecA* stimulates the cleavage of the Pol V regu-
latory subunit UmuD to its active UmuD0 form (Shinagawa et al., 1988).
UmuD0 then forms a homodimer, which pairs with UmuC to create a fully
functional Pol V (UmuD0 2UmuC).
In addition to the regulatory roles above, RecA also participates directly
in the DNA-damage avoidance process. In the recombination-mediated
mode of DNA damage tolerance, RecA acts with RecBCD in DNA double-
strand break repair (Kuzminov, 1999) and with the RecFOR complex to
stabilize stalled replication forks (Chow and Courcelle, 2004; Courcelle
et al., 1997; Webb et al., 1997) and to bypass replication blocks by resuming
replication using a newly synthesized homologous template (Courcelle
et al., 1997; Kogoma, 1997). During Pol V–catalyzed TLS, RecA is required
in two distinct ways; namely, translesion synthesis and the stimulation of
nucleotide incorporation (Pham et al., 2002).
In summary, as illustrated in Fig. 1, prokaryotic PRR employs RecA as a
DNA damage sensor via ssDNA binding affinity, as a signal transducer at
both transcriptional and posttranslational levels, and as an effector that
directly participates in homologous recombination, replication restart,
and TLS. Notably, the regulatory role of RecA in prokaryotic PRR involves
the modification of proteins via protease cleavage. In contrast, eukaryotes
have developed a very different and potentially more sophisticated strategy
of PRR regulation; namely, modification by protein conjugation.
III. DNA Postreplication Repair in Eukaryotes

The PRR response in eukaryotes is apparently very similar in overall
strategy to that of prokaryotic cells (see Fig. 1). In each case, the endpoint
is recombination-mediated damage avoidance or TLS to allow replication
past DNA lesions that would otherwise result in cell death. The similarities
end there, however, as the molecular architecture, biochemical activities,
and signaling events in PRR are strikingly different between prokaryotes
and eukaryotes. Because the PRR pathway has been best characterized in
S. cerevisiae, which is the only model to date to illustrate eukaryotic PRR
mechanisms, our discussions of PRR in this chapter will focus on a yeast
perspective.
Classical genetic analysis has led to the convention that PRR consists of
branched pathways that include at least one ‘‘error-free’’ and one ‘‘error-
prone’’ pathway (reviewed in Broomfield et al., 2001). The RAD6 and
RAD18 genes are required for both subpathways, and mutations that
282
PASTUSHOK AND XIAO
Fig. 1. Comparison of the DNA postreplication repair response in prokaryotes and eukaryotes. Shaded blocks indicate functionally
conserved steps between the Escherichia coli and Saccharomyces cerevisiae pathways.
disrupt either gene result in the most severe DNA damage sensitivities of
all PRR genes. RAD6 encodes a ubiquitin-conjugating enzyme (Ubc or E2)
that forms a heterodimer with Rad18, an ssDNA binding protein with
ATPase activity. The error-prone branch consists of Pol (Rev3 þ Rev7)
and the UmuC homolog, Rev1, which are discussed in detail in other
chapters of this book. A parallel branch consists of an E2 complex com-
prises Ubc13-Mms2 and Rad5, a ssDNA-dependent ATPase. Given the fact
that the ubiquitination activity of both Rad6-Rad18 and Mms2-Ubc13-Rad5
complexes is essential for their PRR functions, it is necessary to briefly
review the ubiquitination process. Readers are encouraged to refer to
recent reviews in this field (Hershko and Ciechanover, 1998; Hochstrasser,
1996; Jentsch, 1992; Pickart, 2001).
IV. Ubiquitination
Since the discovery of Ub (Schlesinger et al., 1975) more than 20 years
ago, ubiquitination (Hershko et al., 1983) has become one of the corner-
stones of covalent protein modification. An explosion of research in
the area in the last 10 years has revealed biological roles for ubiquitination
that rival the scope of phosphorylation. Because its traditional and best-
characterized role is a fundamental biological process found in eukar-
yotes, namely, proteasome-dependent protein degradation, it is not
surprising that ubiquitination has such a broad cellular influence.
More recently, however, breakthrough discoveries in the field have revea-
led an even greater depth and versatility, several examples of which are
encountered in the PRR pathway.
In the simplest sense, ubiquitination is a three-step biochemical reaction
that uses Ub, a small, globular, 76–amino acid protein to covalently modify
its targets. As the name implies, Ub is found throughout eukaryotic cells,
and with merely a three–amino acid difference between lower and higher
eukaryotes, it is one of the most conserved proteins in nature (Ozkaynak
et al., 1984). The biochemical reaction is initiated when an ATP-dependent
ubiquitin-activating enzyme (Uba or E1) forms a high-energy thiolester
bond between the C-terminal Gly76 residue of Ub and an internal Cys
residue of the E1. Ub is next transferred from the E1 to form another
thiolester bond, this time with the active-site Cys residue of an E2. The
final step links the C-terminal residue of Ub to a surface "-amino group of
a Lys residue on the target, forming an isopeptide bond that usually, but
not always, requires a ubiquitin-ligase (Ubl or E3) that may itself interact
with Ub.
Although the E1 performs a rather general role and is encoded by a
single or very few genes in the cell, E2s and E3s are responsible for the
versatility and target specificity of ubiquitination. E2s comprise a family of

proteins with a highly conserved core domain of approximately 150 amino
acids. On the basis of various crystallographic data, the core domain forms
a globular / protein with a central -sheet and surrounding -helices
(Moraes et al., 2001; Pickart, 2001; see Fig. 2A). Within this region lies an
obligatory Cys active site that is absolutely conserved among all E2s and
that is housed in a shallow cleft in the center of the protein (VanDemark
Fig. 2. Structure and function of the Ubc13-Mms2 heterodimer. (A) Crystal

structure of the human Ubc13-Mms2 heterodimer at 1.8 Å resolution (Moraes et al.,
2001). Mms2 binds Ubc13 end-on to form a ‘‘T’’ shape. Mms2 adopts a similar / fold
to Ubc13, but without two C-terminal -helices. (B) van der Waals space-fill
representation of the Ubc13-Mms2 surface. Green, Mms2; blue, Ubc13; yellow, active-
site cysteine of Ubc13. Residues undergoing chemical shift on Ub-Ubc13 thiolester
formation, and the Ub-Mms2 noncovalent interaction are shown in tan and rose,
respectively. Arrows indicate channels through which Ub passes during ubiquitination.
Molecular graphics in both (A) and (B) were generated using RasTop software (Valadon,
http://www.geneinfinity.org/rastop/). (C) Ubc13-Mms2-mediated ubiquitination in-
volves Ubc13 thiolester formation with ‘‘donor’’ Ub, Mms2 orientation of the ‘‘acceptor’’
Ub, and direct or indirect delivery to the substrate. Within the yeast PRR pathway, it is
believed that Rad5 acts as the E3 and PCNA is the substrate. (See Color Insert.)
and Hill, 2002). As evidenced by multiple E2 crystal structures (e.g., see

Cook et al., 1993; Moraes et al., 2001; Worthylake et al., 1998), the three-
dimensional organization of all E2s is similar and creates a typical Ubc
fold. Although confined to these general similarities, E2 enzymes have the
potential for diversification. Indeed, S. cerevisiae contains only 11 E2s that
are able to covalently attach Ub to various target proteins. The diversity
and specificity are believed to be achieved in several ways. Many E2s are
longer than the core sequence and consist of C- or N-terminal extensions
and are classified as Class II and Class III Ubcs, respectively ( Jentsch,
1992). It is believed that such E2 extensions mediate specific downstream
interactions with E3 proteins or the actual target of ubiquitination (Pick-
art, 2001). More important, the ability of a single E2 to bind more than
one E3 is probably the determining factor in specifying a given substrate,
as the E3s represent the most plentiful group of proteins in ubiquitina-
tion. E3s are structurally diverse and act alone or as part of a multisubunit
complex to fulfill their functions as either active or passive adapters
between the E2 and substrate. To date, only two characteristic domains
have been well defined in some E3 proteins. The HECT (homology to the
E6-AP carboxyl terminus) domain is able to bind Ub in a thiolester-
dependent manner akin to E1 and E2 enzymes (Scheffner et al., 1995).
In contrast, RING finger–containing E3s do not necessarily participate in
conjugation reactions but act as adapters between conjugation enzymes
and their substrates via a RING finger motif (reviewed in Joazeiro and
Weissman, 2000). The discovery of the RING finger was based on its
similarity to the DNA-binding zinc finger motif (Lovering et al., 1993).
Similar to the zinc finger, the RING finger fulfills a structural purpose to
stabilize an otherwise unstructured loop region by creating a scaffold
via intrapeptide binding of zinc cations. The RING finger differs in struc-
ture from the zinc finger in that it has a longer consensus sequence
designated Cys-X2-Cys-X(9–39)-Cys-X(1–3)-His-X(2–3)-Cys/His-X2-Cys-X(4–48)-
Cys-X2-Cys, where X is any amino acid (Saurin et al., 1996), and in its
propensity for binding other proteins (especially Ubcs) instead of DNA.
Several crystal structures of proteins containing RING motifs have been
solved, the most significant of which is the structure of the Ubc-RING
finger heterodimer of UbcH7 and c-Cbl (Zheng et al., 2000).
A single pass through ubiquitination results in the tagging of substrate
by a single Ub moiety (mono-Ub), which in some cases is the desired end
product. Alternatively, poly-Ub chains can be formed if successive reac-
tions take place and an internal lysine of another Ub molecule is used as
the subsequent site of attachment. Poly-Ub chains are most commonly
conjugated via Gly76-Lys48 linkages to provide a characteristic ‘‘flag’’ for
signaling protein degradation by the 26S proteasome (Hochstrasser,
1996). However, the structure of Ub reveals six other surface-

exposed lysine residues (Vijay-Kumar et al., 1985, 1987), and thus a new
and growing field recognizing alternatively linked poly-Ub chains has
emerged. To date, poly-Ub chains made via Lys63 (Spence et al., 2000),
Lys-11 (Baboshina and Haas, 1996), Lys29 (Arnason and Ellison, 1994),
and Lys6 (Wu-Baer et al., 2003) linkages have been reported.
Although the E2s and E3s provide two levels of variability in ubiquiti-
nation, the potential for different poly-Ub conjugates allows a third
and fascinating level of versatility. At a fundamental level, it implies a
mechanistic diversity with regard to enzyme catalysis, as the surface lysines
of Ub are well dispersed. Because E2s are so conserved within their cata-
lytic domain, the ability of one E2 to conjugate Ub through a different
Lys than another E2 indicated that the first E2 either possesses correspon-
ding intrinsic differences or functions along with other, potentially
novel accessory factors. A case for the latter was recently discovered
for the function of the Ubc13-Mms2 complex in PRR and will be discus-
sed below. Another consequence of nonclassical poly-Ub conjugates
is that different conjugates may lead to different physiological sig-
nals. For example, although typical Lys48 poly-Ub conjugates signal for
proteasome degradation, Lys63-mediated poly-Ub conjugates signal
for various cellular processes such as a stress response (Arnason and
Ellison, 1994), mitochondrial inheritance (Fisk and Yaffe, 1999), plasma
membrane protein endocytosis (Galan and Haguenauer-Tsapis, 1997),
ribosome function (Spence et al., 2000), and PRR (Hofmann and Pickart,
1999).
V. Protein Conjugation in PRR

Perhaps the most fascinating aspect of eukaryotic PRR is not simply how
it has adapted to use protein conjugation in place of protein cleavage as in
prokaryotes, but that it does so in ways that have set new precedents and
revealed completely novel mechanisms in DNA repair. The following
sections will describe the molecular framework behind the three protein
conjugation complexes in PRR.
A. Rad6-Rad18
On the basis of the conventional genetic hierarchy of DNA postreplica-
tion repair, as discussed above, Rad6 is considered the hallmark and
starting point for the pathway. RAD6 encodes a multifunctional Ubc
(Jentsch et al., 1987) that, in addition to its roles in DNA repair, functions
in histone modification and N-end rule protein degradation. Because the

E2 activity of Rad6 is required for all its known biological roles (Sung et al.,
1990, 1991), it stands to reason that Rad6 fulfills its distinct functions
through interaction with different E3s or via different Ub conjugations, or
both.
One of these functions is revealed by the ability of S. cerevisiae Rad6 to
create and attach poly-Ub chains to histones in vitro (Sung et al., 1988).
Subsequent in vitro studies confirmed the E2 activity of Rad6 for histones
but went on to suggest atypical poly-Ub chain catalysis (Haas et al.,
1991). Although Rad6 can indeed form poly-Ub chains via Lys6 in vitro
(Baboshina and Haas, 1996), the in vivo evidence remains elusive. Another
curious aspect of Rad6 is its specificity for histones in vitro in the absence
of an E3. Because Rad6 is a Class II E2 with a C-terminal tail that lies
beyond the core Ubc domain, it has been hypothesized that the C-terminal
string of 14 consecutive acidic amino acids provides affinity for positively
charged histones. This view is supported by studies that show that the
deletion of the Rad6 C-terminal tail abolishes its ability to ubiquitinate
histones, but not its DNA repair function (Sung et al., 1988). However, the
role of Rad6 in histone modification has been substantiated in vivo
(Robzyk et al., 2000), and contrary to in vitro studies, Rad6 requires an
E3 RING finger protein, Bre1 (Hwang et al., 2003; Wood et al., 2003).
However, the interplay, if any, between the C-terminus of Rad6 and Bre1
has yet to be elucidated. Also, these studies indicate that the in vivo
modification of H2B by Rad6 is by mono-Ub. Such conjugates are
not sufficient for proteasome degradation and are instead implicated in
stabilization or signaling.
Another early study of ubiquitination by Rad6 involves a process called
the ‘‘N-end rule,’’ in which the in vivo half-life of some proteins is
determined by the nature of their N-terminal amino acids (Bachmair
et al., 1986). The N-end rule is dependent on the E2 activity of Rad6
(Dohmen et al., 1991), and as such, Rad6 catalyzes the formation of typical
Lys48 poly-Ub conjugates (Chau et al., 1989). These tagged proteins are
subsequently degraded by the 26S proteasome. In contrast to histone
modification, the role of Rad6 in the N-end rule has shown a clear and
defined requirement for a RING finger E3 protein, Ubr1, and classical
Lys48 poly-Ub chain catalysis for proteasome degradation.
The multifunctionality of Rad6 is further supported by the fact that
the ubr1 mutation does not affect PRR, whereas the rad18 mutant is
completely defective. Indeed, Rad6 is able to form exclusive complexes
with Rad18 or Ubr1 (Bailly et al., 1994), and the Rad6-Rad18 heterodimer
is required for PRR (Bailly et al., 1997a). The Rad18 protein contains the
aforementioned RING finger motif and has ATPase and ssDNA binding
activities (Bailly et al., 1997b). These functions fit well with a working

model in which Rad18 brings Rad6 into close proximity to ssDNA gaps
so that the ubiquitination of a target protein by Rad6 can initiate the
PRR process. The ssDNA binding activity of Rad18 is consistent with
observations that PRR pathway mutants are sensitive to killing by a broad
range of DNA-damaging agents, which may all potentially lead to a com-
mon replication-blocking end product (Broomfield et al., 2001; Prakash
et al., 1993).
Some insight into the Rad6-Rad18 complex (and Rad6-Ubr1) has been
made possible by the yeast Rad6 crystal structure at 2.6 Å (Worthylake et al.,
1998). As expected from the high sequence homology between E2s, Rad6
adopts the characteristic / fold of the Ubc family and does not contain
structural elements that differ significantly from the other Ubcs. Notably,
the highly acidic C-terminus unique to Rad6 in S. cerevisiae could not be
resolved. Although the Rad6 structure is perhaps not profoundly insight-
ful on its own, it became a valuable tool for visualizing the predicted
interacting regions for Rad18 and Ubr1. Using data derived from Rad6
deletion analysis and subsequent binding studies with Ubr1 (Watkins et al.,
1993) and Rad18 (Bailly et al., 1997), the residues important for interac-
tion in each case were mapped to the Rad6 surface (Worthylake et al.,
1998). Using this method, both Rad18 and Ubr1 were implicated in
interacting with similar surfaces on Rad6. These regions are distal from
the Rad6 active site and indicate an indirect role, if any, for these E3s in
the ubiquitination reaction. This finding underlines that Rad18 and Ubr1
provide roles in defining the biological process for Rad6 and confirms the
observation that Rad6 forms distinct heterodimers with each E3.
These early structure and function studies demonstrate an ability
of Rad6 to catalyze distinct protein modifications; namely, mono-Ub
and poly-Ub chains. As a result, it remained difficult to make inferences
as to the Ub modification by Rad6-Rad18 in PRR. Given the assumption
that both Ubr1 and Rad18 bind the same surface of Rad6, one might
assume that the ubiquitination in PRR is via Lys48 as it is in the N-end
rule. However, it was very recently discovered that the Rad6-Rad18 com-
plex targets proliferating cell nuclear antigen (PCNA) with a single
Ub moiety (Hoege et al., 2002), indicating another level of variability in
Rad6-mediated Ub conjugation by its binding partners.
B. Mms2-Ubc13-Rad5
The second instance of ubiquitin conjugation in PRR involves an atypi-
cal poly-Ub chain and a novel mechanism of catalysis employing a Ubc
enzyme variant (Uev), Mms2. The MMS2 gene was isolated by functional
complementation, and genetic analyses indicate that MMS2 belongs to the

RAD6 pathway (Broomfield et al., 1998). However, unlike rad6 and rad18
mutants, the mms2 mutant displays moderate sensitivity to DNA-damaging
agents but significantly increased spontaneous mutation rates. Although
the increased mutation rate in mms2 is completely abolished by inactiva-
tion of the REV3-mediated TLS pathway, the mms2 and rev3 mutations
exhibit a remarkable synergism with respect to killing by DNA damaging
agents. These observations place MMS2 in an error-free branch of PRR
parallel to the Pol-mediated mutagenesis pathway (Broomfield et al.,
1998; Xiao et al., 1999).
Uevs are similar in sequence to Ubcs but lack the obligatory active site
Cys residue required for Ub conjugation. Uev homologs have been found
in essentially all eukaryotic organisms examined to date, including hu-
mans (Sancho et al., 1998; Xiao et al., 1998). Phylogenetic analysis also
indicates that Uevs have evolved as a distinct class of proteins from Ubcs,
early in eukaryotic evolution (Villalobo et al., 2002). Because the Uev
domain adopts a similar three-dimensional structure to the Ubc (Fig. 2A)
but does not form Ub conjugates, it had been hypothesized that Uevs
function as dominant-negative regulators of ubiquitin conjugation
(Koonin and Abagyan, 1997). Given the epistatic relationship between
rad6 and mms2 in DNA repair, it was suggested that Mms2 functions as an
accessory protein that positively modulates the ubiquitination activity of
Rad6 (Broomfield et al., 1998). However, rigorous in vivo and in vitro
studies failed to demonstrate a physical link between Mms2 and Rad6
(Xiao, W., and Broomfield, S., unpublished observations) and it turned out
that Mms2 forms a stable complex with a different and novel Ubc, Ubc13.
The physical interaction between Ubc13-Uev/Mms2 was demonstrated
by using Ubc13 and Mms2 as bait for copurification (Hofmann and
Pickart, 1999) and yeast two-hybrid screens (Brown et al., 2002), respec-
tively. More significantly, it was discovered that Ubc13 is the only known
Ubc capable of Lys63 poly-Ub chain assembly, and that its associated Uev is
absolutely required for this process (Hofmann and Pickart, 1999). Indeed,
deletion of UBC13 in yeast cells results in phenotypes indistinguishable
from those of mms2 mutants (Brusky et al., 2000), thus placing UBC13 in
error-free PRR together with MMS2.
Because Ubc13 consists solely of the core Ubc domain but catalyzes
atypical poly-Ub chains via Lys63, the role for Mms2 as a positive regu-
lator via its physical interaction with Ubc13 can be envisioned. Indeed,
both yeast (Hofmann and Pickart, 1999) and human (McKenna et al.,
2001) Ubc13-Mms2 form a 1:1 stable heterodimer with binding affinities
(Kd) of approximately 400 (Ulrich, 2003) and 50 nM (McKenna et al.,
2003; Ulrich, 2003), respectively. The role of Mms2 in Ubc13-mediated
polyubiquitination is revealed using two-dimensional 1H-15N HSQC (het-

eronuclear single nuclear quantum coherence) such that an ‘‘acceptor’’
Ub for Lys63 attachment is bound in a noncovalent manner by Mms2. This
finding leads to a model in which Mms2 orients the acceptor Ub via
noncovalent contacts so that its Lys63 is made available to the ‘‘donor’’
Ub of the Ubc13 thiolester (McKenna et al., 2001 and Fig. 2C). The
structural basis of the Ubc13-Mms2 complex and its unique role in
Lys63 polyubiquitination is further elucidated by crystallographic analysis
of yeast (VanDemark et al., 2001) and human (Moraes et al., 2001) com-
plexes. Interestingly, despite the typical Ubc folds adopted by each pro-
tein, the heterodimer is asymmetrical and is formed with Mms2 binding
Ubc13 in an ‘‘end-on’’ or ‘‘T-shaped’’ manner (Fig. 2A).
The interface in each of the yeast and human Ubc13-Mms2 structures is
almost completely conserved and buries nearly 1500 Å of solvent-accessible
surface area. The hydrophobic pocket generated by Ubc13 is not con-
served in other E2s but is highly conserved within Ubc13s from different
organisms. Site-specific mutations of UBC13 and MMS2 that result in
amino acid substitutions of some of the highly conserved residues within
this hydrophobic pocket severely affect or abolish Ubc13-Mms2 complex
formation (Pastushok, L., Moraes, T. F., Ellison, M. J., and Xiao, W.,
unpublished observations; VanDemark et al., 2001).
Perhaps the greatest contribution of the Ubc13-Mms2 crystal structure
analyses is the insight into the basis of Lys63-mediated poly-Ub chain
formation. Notably, both studies identified three main channels or clefts
converging on the Ubc13 active site, through which Ub makes contact or
passes during Lys-63 Ub conjugation (Fig. 2B). Channel I refers to the
typical E2 active site at which the donor Ub forms a thiolester with Ubc13.
Because the Ubc13 structure does not appreciably change on binding
Mms2, it has been inferred that Channel I behaves relatively independent
of Mms2. In contrast, the remaining two channels for Ub interaction are
dependent on the asymmetrical dimerization of Ubc13-Mms2. Channel II
comprises the necessary link between donor and acceptor Ub and provides
the molecular framework for Lys63 poly-Ub chain assembly. Various dock-
ing experiments indicate a common mechanism whereby Mms2 positions
the acceptor Ub within a concave surface such that only the Lys63 residue
is made available for conjugation by the Ubc13 active site–bound donor
Ub. The last identifiable channel to converge on the Ubc13 active site is
also only apparent after Ubc13-Mms2 dimerization. Channel III has been
speculated to be an ‘‘outgoing’’ path for Lys63 Ub conjugates and follows
in a relatively opposite direction to Channel II. Importantly, structures of
the Ubc13-Mms2 heterodimer and the predicted tetramer (Ub-Ubc
13-Mms2-Ub) both indicate that the surface of Ubc13 at the end of
Channel III remains completely available (McKenna et al., 2003). This

exposed region corresponds to the surface required for an E2–E3 interac-
tion, as evidenced by crystal structures of UbcH7 bound to the RING
finger E3, c-Cb1 (Zheng et al., 2000) and the HECT domain E3, E6-AP
(Huang et al., 1999). Although direct experimental evidence to support a
function for Channel III in PRR is currently unavailable, the observation
of a physical interaction between Ubc13 and the RING finger protein
Rad5 (Ulrich and Jentsch, 2000) indicates functional relevance in vivo.
RAD5 was genetically placed within the PRR pathway ( Johnson et al.,
1992) and was also identified as an antimutator, rev2 (Lawrence, 1982). In
addition to its RING finger motif, Rad5 contains a ssDNA-dependent
ATPase domain ( Johnson et al., 1992) and Swi2/Snf2 homologous do-
mains important for chromatin remodeling (Richmond and Peterson,
1996). Therefore, like the Rad6–Rad18 complex, the Rad5–Ubc13 inter-
action provides the framework for another instance of Ub conjugation in
PRR at sites of damaged DNA. Indeed, two-hybrid and in vitro coimmu-
noprecipitation experiments revealed a physical interaction between Rad5
and Ubc13 (Ulrich and Jentsch, 2000). Functional significance for the
interaction was demonstrated with a combination of in vivo localization
and cross-linking experiments that showed nuclear localization of Ubc13-
Mms2 after DNA damage and chromatin association of Rad5-Ubc13,
respectively. Substitution of critical residues within the Rad5 RING finger
abolishes its binding to Ubc13 (Ulrich, 2003; Ulrich and Jentsch, 2000).
To support these findings, the same study revealed specific residues of
Ubc13 that mediate interaction with Rad5, and such residues are situated
at the distal end of Channel III. Therefore, it may be hypothesized that
the Rad5 RING finger plays an active role as an E3 in Ubc13-Mms2 Lys63
poly-Ub conjugation.
C. Ubc9-Siz1
The third and most recent finding of protein conjugation in PRR
does not use Ub, but a small Ub-like modifier (SUMO). SUMO is
the best-studied example of a group of Ub-like proteins (Schwartz and
Hochstrasser, 2003) that can be attached to targets for posttranslational
modification in a manner reminiscent of ubiquitination. SUMO-linked
proteins are involved in a wide variety of cellular processes. Sumoylation is
unique to and conserved throughout eukaryotes and shares similar enzy-
mology to ubiquitination. Although the SUMO molecule shares only
18% similarity with Ub, it adopts a Ub-like fold with conserved position-
ing of C-terminal residues for isopeptide bond formation (Bayer et al.,
1998; Sheng and Liao, 2002). Notably, a protruding flexible N terminus
extends beyond the region of Ub homology, and the charged surface of

SUMO is markedly different than Ub. Furthermore, residues of the Ub
moiety responsible for poly-Ub formation are correspondingly absent
from SUMO (Bayer et al., 1998). The disparity between SUMO and Ub
molecules is indicative of a functionally analogous but distinct set of
sumoylation enzymes. Indeed, the only known SUMO-conjugating enzyme
to date was originally identified as the ubiquitin-conjugating enzyme, Ubc9
(Seufert et al., 1995), but was subsequently demonstrated to conjugate
SUMO instead of Ub (Desterro et al., 1997; Johnson and Blobel, 1997;
Schwarz et al., 1998).
Although the overall strategy of sumoylation is comparable to ubiquiti-
nation, a notable exception is that substrates are almost always tagged with
a single SUMO moiety that does not signal for degradation. Also, the
crystal structure of a Ubc9-substrate interaction (Bernier-Villamor et al.,
2002; Rodriguez et al., 2001) and various in vitro experiments indicate that
target identification by Ubc9 can often occur independent of an E3.
A further divergence from ubiquitination is evidenced by a consensus
sequence found in the target proteins that is commonly used as the site
of SUMO attachment (Rodriguez et al., 2001; Sampson et al., 2001).
The emergence of Ubc9 in PRR was not brought about by direct studies
of DNA repair but through a survey for proteins modified by SUMO in
S. cerevisiae. Immunopurification of SUMO–protein conjugates revealed
PCNA as a novel target of sumoylation, and by biochemical association,
Ubc9 was implicated as the functional E2 (Hoege et al., 2002). Further-
more, the same study identified that SUMO modification of PCNA is
mediated by Siz1, a SUMO E3 previously identified as a factor required
for sumoylation of yeast septins (Takahashi et al., 2001a,b). Interestingly,
as Siz1 contains a zinc-binding RING finger–like domain, the Ubc9-Siz1
relationship represents the third protein-conjugating complex in PRR
mediated by a RING finger E3s.
VI. Postreplication Repair via Covalent

Modifications of PCNA
Because previous studies demonstrated that both Rad6 and Ubc13
ubiquitination activities are required for their PRR functions, it is ex-
pected that the covalent modification of one or more critical targets by
these two ubiquitination complexes signals for their respective PRR activ-
ities (Broomfield et al., 2001). A recent study has clearly pointed to PCNA
as such a critical target (Hoege et al., 2002).
As discussed elsewhere in this book, PCNA (or Pol30 in S. cerevisiae) is an
essential component of the eukaryotic replication machinery. The most
notable function of PCNA occurs while bound to DNA polymerases (Pol

or Pol"), whereby a PCNA trimer encircles DNA and acts as a sliding-clamp
to provide processivity during replication (Paunesku et al., 2001). In
addition, PCNA is involved in other forms of DNA metabolism such as
nucleotide excision repair, base excision repair, mismatch repair, and
postreplicative processing (Kelman and Hurwitz, 1998; Paunesku et al.,
2001). The involvement of PCNA in PRR was originally implicated
through the characterization of a mutant allele of POL30, pol30-46, which
conferred ultraviolet sensitivity (Ayyagari et al., 1995). Genetic analysis of
pol30-46 placed POL30 within the RAD6 pathway (Torres-Ramos et al.,
1996), and pol30-46 appears to be defective in the error-free branch of
PRR (Xiao et al., 2000).
Discovery of PCNA as the target for covalent modification by PRR
proteins was somewhat inadvertent. In screening for new SUMO sub-
strates, Hoege et al. (2002) found that PCNA can be sumoylated. By
systematically mutating each of the 18 Lys residues of PCNA to Arg, it
was revealed that S. cerevisiae PCNA could be monosumoylated at two Lys
residues; namely, Lys127 and Lys164. The authors noted that the promi-
nent site for SUMO conjugation is Lys164, which is conserved within
eukaryotes (Fig. 3). It was observed that SUMO modification of PCNA
increases during S phase and that treatment of cells with excessive DNA
damage resulted in a marked increase in sumoylated PCNA. In contrast,
moderate DNA damage resulted in additional PCNA-specific bands that
persist in ubc9-1 mutants that are defective in SUMO conjugation. Inter-
estingly, pull-down experiments revealed the novel PCNA species to be
conjugates containing one to more than four Ub molecules. Furthermore,
Fig. 3. Alignment of partial PCNA amino acid sequences from various model
eukaryotic organisms. Shaded residues match the consensus. Sc, Saccharomyces cerevisiae;
Sp, Schizosaccharomyces pombe; Dm, Drosophila melanogaster; Ce, Caenorhabditis elegans; At,
Arabidopsis thaliana; Mm, Mus musculus; Hs, Homo sapiens. Lys127 and Lys164 residues of
S. cerevisiae (indicated with arrows) are modified by SUMO only, or Ub and SUMO,
respectively.
the Ub conjugates were induced only after DNA damage, and the site of
Ub attachment is identical to that of SUMO at Lys164.
Because Ub-PCNA is dependent on DNA damage and PCNA was genet-
ically linked to PRR (Torres-Ramos et al., 1996), it was expected that one
of the Ubc complexes in the PRR pathway might be responsible. Indeed,
covalent modification of PCNA was completely abolished in rad6 mutants,
whereas sumoylation was unaffected. Surprisingly, mutants in the error-
free PRR genes UBC13, MMS2, and RAD5 led to the disappearance of poly-
Ub conjugates without affecting the mono-Ub species. It can be thus
deduced that monoubiquitination of PCNA is performed by the Rad6-
Rad18 complex, whereas Ubc13-Mms2-Rad5 is responsible for polyubiqui-
tination of PCNA. Indeed, poly-Ub conjugates of PCNA were absent in
yeast cells solely expressing Lys63-mutated Ub. Therefore, the poly-Ub
conjugates are attributed to the rare Lys63 conjugation activity of
Ubc13-Mms2.
To further explore the mechanisms behind PCNA modification, Hoege
et al. (2002) sought to demonstrate direct physical interactions between
PRR proteins and PCNA. Using the yeast two-hybrid assay, the authors
identified interactions between PCNA and the two RING finger proteins,
Rad18 and Rad5. Similarly, Ubc9 was also shown to associate with Rad18
and Rad5, as well as PCNA. Considering their ssDNA-binding affinities, the
new interactions involving Rad18 and Rad5 can be seen as physical links
between the substrate (ssDNA), conjugation machinery (Rad6, Ubc13-
Mms2, and Ubc9) and target (PCNA) of PRR. Taking into account an
earlier report that Rad18 and Rad5 can each form homo- and heterodi-
mers (Ulrich and Jentsch, 2000), a large multisubunit conjugation com-
plex and its biological effects through covalent modification of PCNA can
be predicted (Fig. 4). Indeed, characterization of the pol30-K164R mutant
phenotypes and its genetic interactions with other members in the RAD6
pathway supports the above proposed model.
Because SUMO and Ub converge on the same conserved residue of
PCNA, it is attractive to speculate that each conjugate acts as a mutually
exclusive switch among different replication and PRR modes. However,
although conjugates of Lys63 poly-Ub chains on PCNA can be firmly
rooted in error-free PRR via Ubc13-Mms2, the interplay and function of
mono-Ub and sumoylation requires further investigation. A recent study
(Stelter and Ulrich, 2003) attempted to address this issue through exten-
sive genetic analysis, and it was found that although mono-Ub of PCNA
activates TLS mediated by both Pol and Pol, the effects of PCNA
sumoylation appear to be more complicated. Sumoylation of PCNA con-
tributes to the extreme sensitivity of rad6 and rad18 mutants and partially
affects Pol-mediated spontaneous mutagenesis. It is argued that the
Fig. 4. Covalent modification by Ub or SUMO modulates PCNA function and

leads to different PRR pathways. Current observations are consistent with a model
that unmodified and sumoylated PCNA functions in DNA replication. Mono- and
Lys63-polyubiquitinated PCNA functions in error-prone and error-free damage
tolerance, respectively. Solid circle with ‘‘s,’’ SUMO; open circles with ‘‘u,’’ Ub. This
model is adapted from Stelter and Ulrich (2003).
sumoylation of PCNA must play a more critical role than its mutant
phenotypes currently offer. One possibility is that the sumoylation of
PCNA acts as regulatory antagonist in PRR by competing with ubiquitina-
tion at Lys164. It should be noted that although Lys127 conforms to the
postulated SUMO conjugation consensus sequence, is sumoylated and
appears to play a minor role in PPR, the residue corresponding to
Lys127 is only found in S. cerevisiae. In contrast, Lys164 is invariable among
PCNAs from various model organisms (Fig. 3).
VII. Functional Conservation of Eukaryotic

Postreplication Repair
Although the genes and enzymology in PRR are rather different in
eukaryotes and prokaryotes, there is sufficient evidence indicating that
the PRR process is highly conserved within eukaryotes, from yeast to
human. First, almost all yeast PRR proteins have apparent homologs in
higher eukaryotes (Table I). Furthermore, most, if not all, of the proteins
listed have retained the functional domains or motifs that are seen in
S. cerevisiae PRR proteins. The only exception is RAD5, which does not have
a true homolog based on protein sequence analysis alone in higher
Table I
Sequence Conservation of Eukaryotic PRR Proteins
Protein Protein name or

(S. cerevisiae) Organism accession number % Identity
Rad6 H. sapiens E2a, E2b 69, 69

M. musculus Ube2a, Ube2b 69, 68
A. thaliana Ubc2 65
D. melanogaster Dhr6 68
C. elegans Ubc-1 61
S. pombe Rhp6 77
Rad18 H. sapiens Rad18 14
M. musculus Rad18 15
A. thaliana n/a —
D. melanogaster n/a —
C. elegans n/a —
S. pombe NP_595423 23
Ubc13 H. sapiens Ubc13 68
M. musculus Ubc13 68
A. thaliana Ubc13a, Ubc13b 66, 65
D. melanogaster Bendless, NP_609715 70, 68
C. elegans Ubc13 66
S. pombe Ubc13 70
Mms2 H. sapiens Mms2, Uev1a 47, 46
M. musculus Mms2 47
A. thaliana Uev1a, Uev1b, Uev1c, Uev1d 53, 51, 53, 52
D. melanogaster NP_647959 46
C. elegans Uev1 45
S. pombe Mms2 65
PCNA H. sapiens PCNA 35
M. musculus PCNA 35
A. thaliana PCNA 39
D. melanogaster PCNA 35
C. elegans PCNA 38
S. pombe PCNA 45
PRR proteins from S. cerevisiae were applied to BLAST analysis, and the highest
scoring homologs from various model organisms were included in the table and shown
with percentage amino acid sequence identity. n/a, not applicable (no sufficient
homology found). Note: BLAST search with Rad5 failed to identify sufficient sequence
homologs from any organisms examined in the database to date.
eukaryotes. Nevertheless, several potential RAD5 functional homologs are

currently under investigation. It is also interesting to note that in some
cases (e.g., RAD6 and MMS2), more than one homolog has been identified
in the mammalian genome (Table I). Second, several genes isolated from
higher eukaryotes are found to be capable of functionally complementing
their corresponding PRR defects in budding yeast (Ashley et al.,

2002; Koken et al., 1991; Xiao et al., 1998). Third, in a few limited cases,
experimental inactivation of mammalian PRR genes in a cell line or in
an animal model results in phenotypes reminiscent of the corresponding
yeast mutants (Li et al., 2002; Roest et al., 1996; Tateishi et al., 2000).
Last but not least, the Lys164 residue in PCNA appears to be conserved
in all eukaryotic organisms examined so far (Fig. 3), indicating that
the mechanism of covalent modification of PCNA in PRR has been
conserved throughout evolution. One can infer from this analysis that
postreplicative DNA repair is an important mechanism for maintaining
genome stability, comparable to other well-characterized repair mechan-
isms such as nuclear excision repair, base excision repair, recombination
repair, and mismatch repair. Certainly, more research needs to be done
in multicellular model organisms and in human cells to determine the
similarities and differences in PRR and to see whether defects in this
pathway lead to human diseases.
VIII. Future Directions

The mechanistic studies of PRR described above have been instrumen-
tal in bridging genetic data with the biochemical activities of various
proteins involved in PRR and allow the development of various elegant
models of the PRR pathway (Hoege et al., 2002; Stelter and Ulrich, 2003).
Although genetic approaches will continue to provide invaluable insights
into the PRR pathway, we anticipate an exciting era in our understanding
of PRR through biochemical characterization, leading to the in vitro
reconstitution of PRR activity. Such assays have become instrumental in
studying other repair processes such as DNA excision repair (Kubota et al.,
1996). Because reconstitution experiments are often performed with
highly purified components under well-defined conditions and in a
piece-by-piece manner, they can help to identify the minimal require-
ments of a particular process. Potentially, the precise order of molecular
and regulatory events can also be elucidated.
It is envisioned that three key steps lead toward an ultimate in vitro
reconstitution of PRR. The first would be the in vitro covalent modification
of PCNA by Ub- and SUMO-conjugating complexes and is justified from
several perspectives. First, most of the proteins (or derivatives thereof )
involved in these modifications have been successfully purified. Notably,
many of these proteins retain their functions after expression in prokary-
otic systems, in which contaminating elements of ubiquitination and
sumoylation reactions do not exist. Second, the components and cofactors
required for Ub and SUMO conjugation have been well documented, and
such in vitro reactions are commonplace. Finally, the identification of

PCNA as a target of covalent modification provides a single point in the
pathway that can be easily monitored. However, whether other unidenti-
fied players still exist en route to PCNA modification remains to be
elucidated but can be addressed through this investigation. It is noted
that Ub or SUMO conjugation to PCNA is probably a regulatory element
and does not likely result in the direct processing DNA. Although the
downstream effects of mono- and Lys63 polyubiquitination of PCNA are
translesion synthesis and recombination or template switching, respective-
ly, the molecular determinants that recognize the conjugates are currently
unknown. This study will lead to the second and more challenging task;
namely, to understand how these conjugation states of PCNA signal the
physiological switch between error-free and error-prone PRR. Ultimately,
the reconstitution of PRR in vitro may involve a series of reactions from the
recognition of the DNA lesion substrate to the use of different strategies to
bypass the lesion, with or without an undamaged homologous chromatid
as a template. Two working models have been proposed (Fig. 5) to
account for possible molecular events in response to Lys63 polyubiquitina-
tion of PCNA by error-free PRR proteins; however, neither has been
confirmed or ruled out because of a lack of proper assays. In this regard,
the reconstitution of PRR activity may pose the greatest challenge in the
field of DNA replication and repair.
Another difficulty concerning in vitro reconstitution of PRR is that
although some of the regulatory mechanisms have now become clear,
our understanding of several other aspects is still in its infancy. In our
opinion, the following issues are pertinent to our understanding of how
PRR is regulated in eukaryotic cells and need to be addressed.
First, one of the regulatory components of PCNA conjugation is its
sumoylation, which presumably competes with Rad6-Rad18-mediated
monoubiquitination and is involved in promoting spontaneous mutagen-
esis (Stelter and Ulrich, 2003). SUMO conjugation to PCNA is increased
during S phase and can also be induced by severe DNA damage (Hoege
et al., 2002); however, the biological significance of this process is un-
known. Interestingly, the DNA helicase Srs2, which serves as a molecular
switch between PRR and recombination (Barbour and Xiao, 2003; Schiestl
et al., 1990), is also cell-cycle regulated (Liberi et al., 2000). Because the
entire PRR activity is dependent on functional SRS2 (Broomfield and
Xiao, 2002; Ulrich, 2001), the relationship between PCNA sumoylation
and Srs2 activity needs to be further investigated.
Second, as discussed before, Rad5 contains predicted structural motifs
indicative of involvement in chromatin remodeling. This unique feature
of Rad5 among PRR proteins may explain additional phenotypes of its
Fig. 5. Two alternative models for error-free PRR via recombinational processes.
(A) A strand exchange model, and (B) a template switching model. Both models
propose that progression of leading strand synthesis in the presence of replication-
blocking DNA damage (represented by a triangle) requires the association of the
two nascent DNA strands, followed by resolution of the intermediary structure via
(A) cleavage of the Holliday junction or (B) reverse branch migration. Adapted from
Broomfield et al. (2001).
mutants compared with those of ubc13/mms2. Indeed, characterization of a

histone H2B mutation htb1-3 points to possible links between Rad5
and histone modification (Martini et al., 2002). Also, the ubiquitination
of H2B by Rad6 is required for optimal mitotic cell growth and meiosis
(Robzyk et al., 2000). Whether chromatin remodeling through histone
modification plays a role in PRR remains to be determined.
Third, it has been previously demonstrated that while Rad6 and Rad18
are nuclear proteins, Ubc13 and Mms2 are largely cytosolic and are
translocated into the nucleus in response to DNA damage (Ulrich and
Jentsch, 2000). The mechanism of this regulation is currently unknown
and needs to be investigated.
Fourth, although RecA in E. coli cells plays a critical role in the tran-
scriptional regulation of SOS regulon genes in response to DNA damage, a
similar phenomenon involving the Rad6–Rad18 complex has not been
reported. Instead, cell-cycle checkpoint genes appear to play a similar role
in eukaryotic cells (Bachant and Elledge, 1998). It would be interesting to
know whether the central regulatory proteins in PRR such as Rad6 and
Rad18 are involved in gene regulation in response to DNA damage.
Fifth, because the currently known modifications of PCNA are on the
same Lys residue and do not signal for degradation, it can be inferred
that a reversible process may play an important role in the restoration of
eukaryotic PRR. Although it is known that desumoylation of PCNA proba-
bly requires Ulp1 (Hoege et al., 2002), a Ub protease involved in deubi-
quitination of PCNA has not been reported. Similar to the negative
feedback by the LexA repressor in the E. coli SOS response (Fig. 1), the
recovery from DNA damage in eukaryotes might result in the concomitant
sumoylation of PCNA to inhibit PRR.
IX. Conclusions
Through the above analysis, it is conceivable that both prokaryotic
and eukaryotic organisms employ a centrally controlled postreplicative
survival mechanism to deal with ssDNA gaps left by replication blocks
during DNA synthesis. In bacteria such as E. coli, the homologous recom-
binase RecA serves as a coprotease to fulfill a regulatory role at the
posttranslational level. Its targets include a transcriptional repressor and
a nonessential DNA polymerase. In eukaryotes, it is the E2–E3 ubiquitina-
tion complex Rad6–Rad18 that plays the central regulatory role by
controlling TLS and an error-free mechanism of lesion bypass, presumably
via synthesis of gapped DNA using a homologous chromatid or homolo-
gous chromosome. Although the only currently known target is PCNA,
other targets may be encountered. The discovery of novel signal transduc-
tion mechanisms in PRR through sequential modification of PCNA by two
E2–E3 ubiquitination complexes sets an important milestone in the
research of eukaryotic DNA repair. Notably, the Rad6–Rad18 complex
bridges monoubiquitination of PCNA with error-prone PRR, whereas
Ubc13-Mms2-Rad5 ties novel Lys-63 polyubiquitination of PCNA with
error-free PRR. In addition, another level of sophistication is revealed by
the sumoylation of PCNA by Ubc9-Siz1, which underlines an elegant
interplay between DNA repair and replication.
Acknowledgments
We thank Michelle Hanna for proofreading the manuscript and other laboratory members
for valuable comments. This work is supported by the Canadian Institutes of Health Research
operating grants (OP-38104 and MOP-53240) to W.X. and a Natural Sciences and
Engineering Research Council of Canada postgraduate fellowship to L.P.
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SOMATIC HYPERMUTATION: A MUTATIONAL PANACEA
By BRIGETTE TIPPIN, PHUONG PHAM, RONDA BRANSTEITTER, AND

MYRON F. GOODMAN
Departments of Biological Sciences and Chemistry,

University of Southern California,
Los Angeles, California, 90089
I. Generation of Antibody Diversity . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 307

II. Somatic Hypermutation . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 313
A. Discovery of a Role for AID . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 313
B. Separation of AID activities for SHM and CSR . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 316
C. Initiation of SHM and CSR by AID . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 317
D. Some Like It Hot—Biochemical Aspects of AID . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 318
E. Mutational Models Depicting Two-Phase Somatic Hypermutation . . . .. . . . . . 319
F. Involvement of MMR and BER . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 321
G. Regulation of AID Expression in SHM and Its Importance in
Cancer Avoidance . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 323
III. Apobec Protein Family . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 323
A. Apobec-1. . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 324
B. Antiretroviral Activity of Apobec3G, G ! A Hypermutation in HIV
and Other Retroviruses. . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 325
IV. Biochemical Perspective. . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 326
References . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . 327
I. Generation of Antibody Diversity

DNA damage, be it chromosomal breaks or base alterations leading to
erroneous coding information, are extremely harmful when occurring
randomly within the genome, but when controlled and targeted, the
outcome can be extremely beneficial to the organism’s survival and
overall fitness. Nature has harnessed the power of mutagenesis for pro-
ductive measures most prominently during the generation of antibody
diversity in the immune response of vertebrates. Humans are born with
a subset of genes dedicated for the production of B-cell antibodies and
T-cell receptors, the immunoglobulin genes. To achieve the tremendous
diversity necessary to recognize a myriad of foreign invaders to the
body, an elaborate series of mechanisms has evolved to convert the
original germ-line set of immunoglobulin genes into millions of variants.
Antibody variation occurs for two main purposes. The first is to allow
recognition and binding of antigen that is achieved through V(D)J

308 TIPPIN ET AL.
recombination and somatic hypermutation (SHM). The second is to allow

antigen binding sites that are created to be expressed with the eight
different constant region genes so that they can mediate many different
effecter functions throughout the body by a process called class-switch
recombination (CSR).
V(D)J recombination is the first step in generating antibody diversity
and occurs throughout early B-cell development in the bone marrow.
This specialized form of recombination introduces site-specific double-
stranded DNA breaks between multiple germline copies of V, D, and J
segments in an immunoglobulin gene and subsequently re-assembles the
fragments to produce transcriptionally competent heavy- or light-chain
genes (Fig. 1). A functional antibody is composed of four distinct immu-
noglobulin polypeptides: two heavy chains (IgH), and two light chains of
either the lambda (Ig1) or kappa (Igk) type. Diversity is achieved by the
random choice of which V, D, and J segments are rejoined to one another
and also through errors that are introduced at the junction sites before
the breaks are sealed (reviewed in Gellert, 2002). Transcription and
translation of functional heavy- and light-chain genes that have successful-
ly undergone V(D)J recombination are essential for proper progression
through B-cell developmental stages. If improper rejoining occurs in
either heavy or light chains, the cell will not receive the proper signals
and thus undergo apoptosis. T-cell-receptor (TCR) genes also undergo
V(D)J recombination in T cells in the thymus (Krangel, 2003; Strominger,
1989).
Initiation of V(D)J recombination begins by the action of site-specific
recombination-activating genes (RAG1) and RAG2 (Mombaerts et al., 1992;
Oettinger et al., 1990; Schatz et al., 1989; Shinkai et al., 1992) in a complex
with high-mobility group (HMG) 1 proteins (Sawchuk et al., 1997; van
Gent et al., 1997), which introduce double-stranded breaks at recombina-
tion signal sequences (RSS) adjacent to the multicopy V, D, and J
segments (Lieber et al., 2003). Conversion of break points into hairpins
by the complex protects the ends from rejoining prior to removal of
unwanted gene segments. Next, Ku70/80 heterodimer (Casellas et al.,
1998; Manis et al., 1998) binds to the ends, possibly displacing the RAG
complex, and a different complex made up of Artemis (Moshous et al.,
2001) and the DNA dependent protein kinase catalytic subunit (DNA-
PKcs) (Blunt et al., 1995; Kirchgessner et al., 1995; Peterson et al., 1995) is
recruited by Ku70/80 to open the hairpinned V, D, or J ends (Ma et al.,
2002). Artemis-DNA-PKcs acts as a nuclease, trimming the ends and in-
creasing the diversity at the junctions (Ma et al., 2002). At this intermediate
step, terminal deoxynucleotidyl transferase (TdT) (Gilfillan et al., 1993;
Kohler and Milstein, 1975; Komori et al., 1993), can add extra nucleotides
SOMATIC HYPERMUTATION
Fig. 1. High-affinity antibodies in mice and human are generated by V(D)J joining and subsequent diversification by somatic
hypermutation (SHM) and class-switch recombination (CSR). The primary antibody repertoire is generated during B-cell development
by site-specific recombination resulting in the fusion of germline V, D, and J gene segments. On encounter with antigens, the V(D)J
309
rearranged genes further undergo SHM and CSR to generate high-affinity antibodies. CSR selection to downstream constant domains
310 TIPPIN ET AL.
in a template-independent manner to increase diversity at the junctions,

but TdT’s contribution is limited by its restricted expression in B cells at
the early pro-B cell developmental stage (Li et al., 1993). During the late
pre-B and immature B stages of development, DNA polymerase (mu)
might be recruited by Ku70/80 (Mahajan et al., 2002) in lieu of TdT to
participate in V-J end-processing at microhomologies (Bertocci et al.,
2003). Finally, the combined action of XRCC4 (X-ray cross complementa-
tion 4) and DNA-ligase-IV link the two coding DNA ends to make a
functional immunoglobulin gene (Gao et al., 1998; Grawunder et al.,
1998; Li et al., 1995; Taccioli et al., 1993).
Following the completion of V(D)J recombination in both heavy- and
light-chain immunoglobulin genes, the B cell reaches maturity and ex-
presses a complete four subunit antibody on its surface. Mature B cells
migrate from the bone marrow to peripheral lymphoid tissues such as the
lymph nodes, spleen, Peyer’s patches in the gut, or tonsils, where they
await an encounter with foreign antigen. The initial diversity created in
each individual mature B cell during V(D)J recombination provides a wide
range of low-affinity binding capabilities for antigens. Once a positive
interaction has occurred between an antibody on the B cell surface
(e.g., IgM, IgD), signaling from a T helper cell triggers the B cell to form
germinal centers (GCs) in peripheral lymph nodes. In the GC, B cells
bound to antigen will undergo positive selection and rapid cell division to
create more clones. During this proliferation process, the second stages of
diversification begin to take place; CSR to convert antibodies into different
isoforms that can better trap, neutralize, and clear antigens from the body
in the immune response through unique effector functions; and SHM
targeted to the V (variable) region in IgH and IgL genes that improve
binding of antigen (Fig. 1).
CSR occurs in the Ig heavy-chain locus in the C (constant) domain
located downstream of the V(D)J variable region (Fig. 1). Eight different
CH regions encode unique isotypes IgM, IgD, 4 distinct IgG, IgE, or IgA.
Initially, mature B cells translate the IgH locus using the most upstream
exon C to produce IgM that is expressed as a low-affinity membrane
bound and secreted isotype. The expression of the C heavy chain is
accomplished by mRNA splicing. Each of the other six C regions contains
a cryptic promoter and repetitive switch region (S) located 50 of the exon
[ 3, 1, ( 2b and 2a not shown), , or "] allows switching from IgM or IgD to other
the isotypes IgG3, IgG1, (IgG2b and IgG2a not shown), IgA, or IgE. P represents cryptic
promoters adjacent to switch (S) regions, E and E30 are enhancer regulatory regions,
and HS4 is an enhancer binding site within the E30 region important for CSR.
SOMATIC HYPERMUTATION 311
coding sequence. After antigen engagement, one or another of the other

CH exons is brought into the position adjacent to the V(D)J region
(displacing C), converting the antibody to a different isotype that can
be expressed on the surface or be secreted (Fig. 1). The complete
mechanism of CSR is far less well understood than V(D)J recombination,
but some key features and players have been defined. Unlike V(D)J
recombination, CSR only occurs in B-lineage cells, and although CSR
is triggered during antigen-driven clonal expansion in germinal
centers (GCs), the process can also occur in other regions of the body
via T-cell-dependent interactions with CD40 ligand or T-cell-dependent
antigens such as lipopolysaccharide (LPS) in combination with cytokine
activators such as interleukin-4 (IL-4) (Stavnezer, 2000).
CSR is directed initially by transcription from the cryptic promoter 50 of
the exon targeted for isotype switching by unique cellular signals (Manis
et al., 2002a). The B-cell-specific transcriptional enhancer E, located
between the JH and C region (Fig. 1), can stimulate transcription from
the CH promoters, but this enhancer is not absolutely required (Bottaro
et al., 1998) for CSR. A second cluster of enhancers located 30 of the IgH C
region (in particular the HS4 enhancer element) can also interact with the
activated CH promoter (Fig. 1) and appears to serve as the regulatory
region for CSR (Khamlichi et al., 2000). Germline CH RNA transcripts
generated from these promoters will undergo splicing and polyadenyla-
tion, but they are not translated into protein (Stavnezer, 2000; Zhang et al.,
1995). Rather, the transcripts themselves appear to help target CSR ma-
chinery to a particular S region (Bottaro et al., 1994; Harriman et al., 1996;
Jung, 1993; Seidl et al., 1998). S regions can vary greatly in different CH
segments (Fig. 1), with some composed of tandem pentamer repeats and
others having 49-base-pair repeats. The lack of a consensus break point or
fusion sequence makes CSR a region-specific process (Kinoshita et al.,
1999), rather than a site-specific recombination process as seen for
V(D)J recombination.
Various models have been suggested to explain the regional break
phenomenon via higher-order structures in the S-regions. However the
recent discovery of the activation-induced cytidine deaminase (AID)
(Muramatsu et al., 1999) has led to the proposal that CSR is actually
targeted by AID-initiated strand-breaks on single-stranded DNA generated
during germline transcription (Chaudhuri et al., 2003; Yu and Lieber,
2003). Both CSR (Lee et al., 2001) and SHM require transcription (Bachl
et al., 2001; Peters and Storb, 1996) as a precursor step in their mechanism,
and both pathways are abrogated in the absence of AID (Muramatsu et al.,
2000), but the two processes can be differentially induced in response
to unique signals. For example, activation of splenic B cells by LPS leads to
312 TIPPIN ET AL.
CSR and not SHM (Manis et al., 2002b). In the case of CSR, transcription
through an S region would generate single-stranded stretches of DNA on
which AID could deaminate C bases, any of which could be converted into
a single-stranded break by the subsequent action of a uracil N-glycosylase
(UNG) and AP-endonuclease (Rada et al., 2002).
The conversion of single-stranded breaks into double-stranded breaks
for a CSR event and subsequent end-processing by NHEJ component
proteins (Casellas et al., 1998; Manis et al., 1998; Rolink et al., 1996), and
perhaps DNA-PKcs (Bosma et al., 2002; Manis et al., 2002a), would ulti-
mately leave little trace of a consensus motif by the completion of the
repair process. The action of AID on any available C in a single-stranded
piece of DNA could help explain the randomness of breakpoints seen. In
addition, R loops that form during CSR might contain short regions of
ssDNA on both strands at the transition between the R loop and the
adjacent dsDNA (Chaudhuri et al., 2003; Yu and Lieber, 2003) on which
AID could trigger a double-stranded break. However, what limits AID
access from acting outside of the S-region during transcription is not yet
understood.
SHM, like CSR, is induced in immunoglobulin genes following antigen
recognition by low-affinity antibody on B cells that migrate to germinal
centers. SHM is specifically targeted to V regions in the immunoglobulin
heavy- and light-chain genes (Lebecque and Gearhart, 1990). Mutations
that result in improved affinity for antigen become positively selected,
eventually leading to the development of ‘‘immunity’’ to the infectious
antigen. The key elements necessary for SHM include active transcription
(boosted by enhancer elements) (Maizels, 1995; Peters and Storb, 1996),
high fidelity (Hi Fi), and error-prone (EP) DNA polymerases, and most
prominently, AID (Fig. 2).
SHM requires AID, with initiation now known to involve AID-catalyzed C
! U conversion on ssDNA (Bransteitter et al., 2003; Chaudhuri et al., 2003;
Dickerson et al., 2003; Pham et al., 2003; Sohail et al., 2003), presumably
within a transcription bubble on the nontranscribed strand. C ! T
transitions are the most commonly observed V-gene mutations and are
notably favored in WRC hotspot sequences (W ¼ A or T, R ¼ purine) that
account for roughly half of all documented SHM mutations (Golding et al.,
1987; Rogozin and Kolchanov, 1992), and will result if U remains in the
DNA and is subsequently copied with a normal Hi Fi pol (i.e., pols or ";
Fig. 3). The schematic description shown in Figure 2 provides a general
picture of enzymes and pathways used during SHM, without committing to
specific molecular models. However, with recent progress made in under-
standing the action of AID, molecular mechanisms can now begin to be
investigated (Fig. 3).
Fig. 2. Integrated model for SHM. Interactions between a transcription factor,

activated by the E enhancer, and transcription complex bound at the promoter (P)
lead to transcription of Ig genes. Transcription-dependent AID-mediated deamination
initiates SHM by converting C to U, preferentially at 50 WRC hotspots in the transiently
created transcription bubbles. Subsequent replication of U-containing DNA by high
fidelity (Hi-Fi) polymerases or by error-prone repair mechanisms ‘‘fix’’ SHM mutations
at 50 WRC and 50 TAA motifs. Single-strand nicks or double-strand breaks have been also
observed in the V-region of Ig genes at 50 WRC hot-spot motifs, caused presumably by an
as yet unidentified endonuclease (Endo). The DNA nicks or breaks may be substrates
for one or more error-prone polymerases (EP pols) to bind and generate mutations. An
example of EP-pols is Pol , which is responsible for mutating A and T sites, primarily
within TAA motifs. MAR designates a matrix attachment region, DJ—the D and J joined
with V-region by V(D)J recombination, C-region—the constant region of Ig genes.
II. Somatic Hypermutation

A. Discovery of a Role for AID
AID was first discovered using a subtractive hybridization screen for genes
activated on induction of CSR(Muramatsu et al., 1999) and has since been
shown to serve highly critical roles in a number of immune-specific processes.
AID expression is restricted to activated B cells (Muramatsu et al., 1999) and
is required for SHM in mice (Muramatsu et al., 2000). AID was also found to
be the culprit in a human immune disorder, Hyper-IgM-2 syndrome, in which
some patients exhibited AID deficiency, resulting in the abolishment of
both CSR and SHM (Revy et al., 2000). Hyper-IgM syndrome (HIGM) is
characterized by normal or elevated serum IgM levels associated with the
absence of IgG, IgA, and IgE isotypes as a result of defective CSR
(Notarangelo et al., 1992). Additional experiments in mice and B cell lines
have verified this AID requirement (Martin and Scharff, 2002; Muramatsu
et al., 2000; Revy et al., 2000).
AID transfection studies have further shown that AID is likely to be
the only B-cell-specific protein required for SHM and CSR. Indeed,
314 TIPPIN ET AL.
transfection of AID into human B-cells at the improper stage of differenti-

ation (Martin and Scharff, 2002), into non-B cells (Martin and Scharff,
2002), and surprisingly even in Escherichia coli (Petersen-Mahrt et al., 2002),
is sufficient to induce hypermutation in all of these cell types, whereas
ectopic expression of AID in fibroblasts induces CSR on artificial sub-
strates (Okazaki et al., 2002). Yet another interesting role for AID is seen in
chicken DT-40 cells, which are unable to carry out the process of gene
conversion in the absence of AID (Arakawa et al., 2002; Flajnik, 2002;
Harris et al., 2002). Gene conversion is an alternative pathway for generat-
ing antibody diversity used by chickens and rabbits in place of SHM.
Inactivation of homologous recombination genes XRCC2, XRCC3, or
RAD51B in chicken DT40 cells also has the same effect of impairing gene
conversion, but in the presence of AID, these cells shift to using the SHM
pathway that normally does not occur in this cell line (Sale et al., 2001).
Because both gene-conversion and SHM pathways are inactivated when
AID is lacking in DT-40, it appears that under normal conditions, when
AID is present, the type of repair protein recruited to a uracil lesion may
be the determinant of the mechanistic outcome, either gene conversion or
SHM. Taken together, the evidence indicates that AID plays a universal
role overseeing entry into all immunoglobulin gene-specific modifications
that increase antibody diversification in a variety of species.
AID was initially thought to function by editing mRNA that encodes an
endonuclease that initiates breaks in V and S regions of Ig genes (Honjo
et al., 2002), but an RNA substrate for AID in vivo has not been identified.
AID, when expressed in E. coli, is able to mutate genes on the bacterial
chromosome (Petersen-Mahrt et al., 2002), the first indication that the
enzyme might act specifically on DNA and not RNA. Biochemical data
subsequently showed that AID deaminates C residues on ssDNA
(Bransteitter et al., 2003; Chaudhuri et al., 2003; Dickerson et al., 2003).
Fig. 3. SHM branched pathway involving the initiator action of AID. Shown at the
left is a region of DNA undergoing transcription (RNA transcript indicated by a curved
line in blue). AID acting on ssDNA exposed in the transcription bubble deaminates C in
the WRC hot spot sequence to initiate the first ‘‘phase’’ of SHM. Subsequent copying of
U by normal replication polymerases will result in a C ! T mutation or alternatively an
error-prone (EP) polymerase can generate a C ! N mutation. A second phase of SHM
(SHM diversification) can occur by using the postreplication mismatch repair (MMR)
pathway to excise the U-G mismatch and generate a repair patch in which a second
deamination by AID can occur, and/or EP synthesis can generate the WA mutational
hotspots. SHM diversification can also occur using the base excision repair (BER)
pathway triggered by the action of uracil glycosylase (UNG) and apurinic/apyrimidinic
endonuclease (APE) to excise uracil and nick the DNA backbone. AID-catalyzed
deamination of C is responsible for initiating both phases of SHM. (See Color Insert.)
316 TIPPIN ET AL.
A transgenic approach has been taken to further investigate whether or

not AID deamination of C ! U was directly involved in SHM by looking at
Uracil N–glyclosylase (UNG) deficiencies. In normal cells, UNG plays a
significant role in removing uracil from DNA via the base-excision repair
(BER) pathway (Lindahl and Barnes, 2001). One would predict that if AID
deaminates DNA, then UNG-deficient mice would fail to remove uracil
from the DNA, and when replicated, this would result in an increased
frequency of transition mutations, especially C!T. UNG-deficient mice
and human cells do exhibit altered mutational patterns with C:G ! T:A
transitions that are significantly greater than transversions (Di Noia and
Neuberger, 2002; Imai et al., 2003b; Rada et al., 2002). In these same
studies, CSR is also inhibited, most likely because of a lack of necessary
double-stranded breaks that likely arise during the normal uracil removal
process. Although other uracil DNA glycosylases such as MBD4, TDG, and
SMUG are present in UNG deficient mice and humans, they do not
appear to compensate for loss of UNG activity at the Ig locus. In fact, in
MBD4 knockout mice, neither SHM nor CSR is perturbed (Bardwell et al.,
2003). Other deficiencies of BER proteins such as DNA glycosylases AAG
and OGG1, Poly (ADP-ribose) polymerase, and Pol fail to alter or impair
SHM (Esposito et al., 2000; Jacobs et al., 1998; Winter et al., 2003). Aside
from the XPV gene (encoding DNA polymerase ), proteins from the
nucleotide excision repair (NER) pathway also fail to have any effect on
SHM ( Jacobs et al., 1998). The effect of the UNG deficiencies therefore
demonstrates that introduction of U into DNA by AID occurs in vivo, and
recapitulates the in vitro biochemical activity found for AID.
B. Separation of AID Activities for SHM and CSR

Mutations in AID suggest that it functions as a multimer of two or four
identical subunits, and that specific domains in each peptide may be
responsible for recruiting factors that mediate the outcome of SHM and
CSR. Expression of a double mutant in the catalytic domain of AID in a
Ramos B cell line has a dominant negative effect on the wild-type AID,
abrogating normal activity (measured by SHM), and argues that com-
plexes must form between mutant and wild-type proteins (Papavasiliou
and Schatz, 2002).
A naturally occurring dominant negative mutant was also found in a
Hyper-IgM2 patient who was determined to have a heterozygous mutation
within AICDA, the gene encoding AID (Kasahara et al., 2003). Typical
Hyper-IgM type 2 patients contain homozygous mutations within AICDA
and lack CSR as well as SHM functions (Revy et al., 2000). There are
alternative forms of the syndrome that reveal possible separation of AID
activity between CSR or SHM pathways. HIGM type 4 patients maintain

SHM activity yet lack CSR (Imai et al., 2003a). Mutations within the
189–198 amino acids of the C-terminal portion of AID produce the HIGM
type 4 phenotype (Barreto et al., 2003; Ta et al., 2003), implying a critical
role for this region during CSR, but an expendable role in SHM. Of
note, the C-terminal mutants also exhibit increased cytidine deaminase
activity compared to wild-type AID when expressed in E. coli (Barreto et al.,
2003). Overall, these results suggest that different domains on AID must
require the interaction of additional factors to specify the pathway of AID
targeting to either SHM or CSR in response to unique cellular signals.
In addition to the naturally occurring mutations within AICDA, three
alternative spliced forms of AID have been found. In Splice variant 1
(SV1), the intron between exon 3 and 4 is retained. In splice variant
2 (SV2), exon 4 is removed. Last, in splice variant 3 (SV3), a short neo-
exon in intron 3 is retained and exons 3 and 4 are spliced out (Greeve
et al., 2003). SV1 and SV2 are expressed during the centrocyte B cell stage,
a time when AID is typically down-regulated in the cell (McCarthy et al.,
2003). In contrast, these same alternatively spliced transcripts (SV1 and
SV2) have also been found in B-cell lymphomas at the centroblast stage,
the normal stage for high-level AID expression (Greeve et al., 2003;
McCarthy et al., 2003; Oppezzo et al., 2003). In the case of the B centroblast
lymphomas, in the combined presence of the splice variants and high-level
wt AID, SHM is not observed (McCarthy et al., 2003).
A possible explanation for these results is that the alternative spliced
forms of AID may act as dominate negative regulators, serving to down-
regulate AID function in healthy centrocytes, repressing SHM and CSR,
but when abnormally expressed in centroblasts, may trigger B cell lym-
phogenesis. The splice variant studies have also revealed even further
evidence of the ability to uncouple CSR and SHM through the function
of AID. In follicular lymphoma B cells, the distinct pattern of SV2 and SV3
expression exhibits intact SHM, but is defective for CSR (Greeve et al.,
2003). In contrast, in another study on chronic lymphoid leukemia (CLL),
cells that express both wt AID and splice variants were found to have
defective SHM but intact CSR (Oppezzo et al., 2003).
C. Initiation of SHM and CSR by AID

Baculovirus-expressed GST-AID fusion protein revealed that not only
does AID act specifically to deaminate C residues in ssDNA in vitro, but
it fails to work on dsDNA, DNA/RNA hybrids or RNA alone (Bransteitter
et al., 2003). The initially cryptic activity of AID was revealed after it
was found that the purified GST-AID was bound to an inhibitory RNA
318 TIPPIN ET AL.
molecule, but the putative biological relevance of this RNA inhibition of

AID is yet to be determined (Bransteitter et al., 2003). Additional studies
using partially purified AID from B cell extracts as well as AID purified
from E. coli also showed deamination of ssDNA in vitro (Chaudhuri et al.,
2003; Dickerson et al., 2003; Sohail et al., 2003).
During activation of SHM and CSR, AID must somehow gain access to
ssDNA, which is postulated to occur through opening of a transcription
bubble in the V region (Fig. 2) or at one of the various CH loci. Evidence to
support this model comes from several experiments. First, AID-dependent
deamination during a prokaryotic RNA polymerase-driven transcription
reaction exhibits a 15-fold preference for the nontranscribed strand (Pham
et al., 2003; Ramiro et al., 2003; Sohail et al., 2003). Second, GST-AID activity
was found to be greatest on a small 9-nt transcription-like DNA bubble
(Bransteitter et al., 2003), and although any connection of this observation
to SHM in the ‘‘real world’’ would obviously be naive, it may indicate a
potential connection to transcriptional targeting. Third, it is well docu-
mented that both CSR and SHM require transcription to proceed
(Maizels, 1995; Manis et al., 2002b; Peters and Storb, 1996), and a tran-
scription bubble constitutes an obvious potential entry point for AID as
transcription generates ssDNA regions on the nontranscribed strand.
Regardless of the specific targeting mechanisms for AID to V regions for
SHM and C regions for CSR, the genetic and biochemical data suggest that
AID deamination on DNA in Ig genes is a critical early step in these
processes.
D. Some Like It Hot—Biochemical Aspects of AID

Attempts to reconstitute the entire SHM process in a test tube are in
their infancy; however, there are already several biochemical observations
involving AID that directly simulate some of the most salient features of
SHM. A hallmark of SHM is that transition mutations are favored over
transversions occurring predominantly in WRC hotspot sequences (in-
cluding GYW on the opposite strand) (Rogozin and Kolchanov, 1992).
Perhaps the strongest biochemical evidence for a direct role of AID on
DNA during SHM was obtained through analysis of the mutational
spectrum for GST-AID on ssDNA that revealed preferential deamination
of dC in WRC hotspot motifs (Pham et al., 2003). AID, acting on ‘‘naked’’
ssDNA, with no additional cofactors, exhibits precisely the same sequence
specificity for C ! U conversions (Pham et al., 2003). The WRC mutability
index, which is defined as the number of times an oligonucleotide
sequence within a segment of DNA contains a mutation divided by the
number of times the sequence is expected to mutate for a mechanism with
no sequence bias, has a value for AID of 1.85 0.26 compared to 1.88
0.59 for SHM in vivo (Pham et al., 2003). This remarkably close agreement
between the biochemical specificity of AID and biological specificity of
SHM indicates that the V-gene targeting at WRC hotspots may depend
primarily, if not exclusively, on AID.
AID also appears to act in a nondistributive manner on naked DNA and
to give rise to a broad clonal distribution of mutations. The muta-
tional spectra study shows that roughly half of lacZ reporter sequences
contained between 2 and 20 mutations per clone, with the remaining half
of the clones containing greater than 20 and up to as many as 80 muta-
tions (Pham et al., 2003). An examination of individual sparsely mutated
clones reveals that deamination of hot-spot C residues could occur a
hundred or more nucleotides apart, with intervening hotspot C residues
remaining untouched. Perhaps AID, which is probably acting as a multi-
mer, can engage one or more of its subunits to bind distal regions along
ssDNA in search of target C residues. In more heavily mutated clones,
clusters of closely spaced Cs, including even adjacent C residues, are
deaminated, with each clustered region generally containing at least one
hotspot sequence. Perhaps these clones demonstrate the potential for
each AID subunit to translocate processively over relatively short distances,
catalyzing closely spaced deaminations in a clustered region allowing
multiple hits by each subunit (Bransteitter et al., 2004).
E. Mutational Models Depicting Two-Phase Somatic Hypermutation

The question arises as to how might mutations be generated sub-
sequent to the action of AID, because although AID can target V-gene
mutations, it cannot generate the entire spectrum observed in vivo. SHM
may best be modeled as two-phase process (Petersen-Mahrt and Neuber-
ger, 2003). The simplest picture describing the first SHM stage would have
AID-catalyzed C deamination at WRC followed by faithful replication of U
by a high-fidelity polymerase, resulting in a C ! T transition (Fig. 3).
Alternatively, a transversion mutation could take place at WRC sites by
copying the same U with an error-prone polymerase instead (Fig. 3).
The model implies that an interaction between AID and RNA poly-
merase during transcription enables AID to load and deaminate Cs on
the nontranscribed strand while confined within a moving transcription
bubble. Recent biochemical data indicate the possibility of a direct inter-
action between AID and RNA polymerase II and RPA (Chaudhuri et al.,
2004; Nambu et al., 2003). Transcription-dependent AID deamination
studies performed in E. coli (Ramiro et al., 2003) and in vitro (Pham et al.,
2003; Sohail et al., 2003) show that deamination is strongly favored on
320 TIPPIN ET AL.
the nontranscribed strand. However, that’s not what happens in vivo,

where V-gene mutations have been found to occur in roughly equal
numbers on both the nontranscribed and transcribed strands (Milstein
et al., 1998), nor can the action of AID alone account for SHM hotspots in
WA motifs.
V-gene mutations can be generated on both strands and further diver-
sified through a second SHM phase in which AID is involved in initiating
the mutational process but not necessarily in targeting the mutations to a
specific site on DNA (Fig. 3). Recent evidence indicates that at least two, or
perhaps more, polymerases are engaged in SHM. Although there appears
to be no change in frequency of SHM in xeroderma pigmentosum
patients lacking pol , P. Gearhart and colleagues (Zeng et al., 2001)
found a measurable reduction in mutations at A:T base pairs and an
increase in mutations at C:G base pairs in V genes obtained from the
peripheral blood lymphocytes of XP patients. Furthermore, analysis of
switched memory B cells in XP-V patients revealed that pol is also an A/T
mutator during CSR, in both the switch region of tandem repeats as well as
upstream of it, thus indicating that the same error-prone translesional
polymerases might be involved, together with AID, in both SHM and CSR
processes (Faili et al., 2004).
These data indicate that pol is specifically an A/T mutator. Using
error-prone pol to copy a gapped DNA construct containing a lacZ
reporter sequence, either alone or aligned in frame with a mouse Ig
light chain transgene, Kunkel and colleagues (Pavlov et al., 2002; Rogozin
et al., 2001), showed that the base-substitution spectra at A-T pairs
correlated with SHM WA hot spot motifs (Rogozin et al., 2001). There
was, moreover, good agreement with mouse data for A ! G substitutions
(but not C ! T substitutions) when error-prone pol was used to copy a
mouse Ig light-chain transgene in vitro (Pavlov et al., 2002). The data also
revealed that avidly mutated WA motifs were often situated close to WRC
motifs (Pavlov et al., 2002).
Based on the close proximity of the two types of hot spot motifs, it is
possible that AID-catalyzed deamination of C residues in WRC could
therefore provide a mechanism for loading an EP pol to copy nearby WA
motifs. Conversion of C ! U by AID, followed by U removal by UNG þ APE
enzymes, results in a nick at the 50 -end of the abasic moiety. If an error-
prone repair polymerase (e.g., pol ) binds and carries out strand displace-
ment synthesis (long-patch BER), then misincorporation of G opposite T
(Fig. 3) will yield A ! G transitions in the nontranscribed strand at WA
hotspots, in accordance with pol ’s mutational specificity (Pavlov et al.,
2002), as shown (in red) in Fig. 3.
In support of the EP pol model, genetic studies with knockout mice

have revealed that two additional EP polymerases, pol and pol , might
participate in SHM. Deficiencies of pol in a Burkitt’s lymphoma cell
line dramatically impair the mutation frequency increase on activation of
these cells, despite the presence of AID, and it was restored by overexpres-
sion of the polymerase (Faili et al., 2002). However, a conflicting report
from mice deficient for pol indicates no change in the frequency of SHM
(McDonald et al., 2003).
The difference between the two studies is yet to be reconciled. Pol can
substitute for pol in a BER in vitro reaction (Bebenek et al., 2001),
indicating that pol could be involved in generating SHM through
error-prone BER repair of G:U mismatches. During short-patch BER,
pol might misinsert nucleotides opposite G templates, generating muta-
tions at G/C sites. In addition, substitution for pol , with a relatively weak
dRP lyase activity, in place of pol could result in a flap substrate (Fig. 3).
Ensuing longer gap-filling synthesis (long patch BER) by error-prone
pol copying nearby TW motifs on the transcribed strand should gene-
rate mutations at A-T pairs (Fig. 3). Pol may also participate in generat-
ing SHM. Knocking out the pol catalytic subunit, REV3, by specific
antisense oligonucleotides impairs B-cell SHM in Ig and BCL6 genes
(Zan et al., 2001). Similarly, transgenic mice, expressing antisense RNA
to a REV3 gene, exhibit decreased frequency of SHM and impaired affinity
maturation (Diaz, 2001).
A simplified overview of the speculative two-phase mutational model
(Fig. 3) is that AID is tacitly assumed to act first by converting C ! U. Then
U may be copied accurately or inaccurately by a high-fidelity or low-fidelity
polymerase, respectively, leading to mutations on the nontranscribed
strand (Fig. 3). A possible scenario to explain the variety of mutations
generated during SHM entails competition between the removal of U
in DNA by BER or MMR and the possibility of direct replication of U in
the DNA by a HiFi or EP pol. Removal of U through BER or MMR,
coupled with the action of EP pols, could then result in mutations at
different sites on the transcribed strand (Fig. 3). Yet another possibility
could be that in contrast to prokaryotic transcription, the transcribed
strand might be susceptible to AID-catalyzed C deamination during
eukaryotic transcription.
F. Involvement of MMR and BER

As illustrated by the model (Fig. 3), postreplicative mismatch repair
also appears to play a role in the SHM process at A/T base pairs. Mice
deficient in the MutS homologs (Msh) 2 or Msh6 have fewer mutations
322 TIPPIN ET AL.
at A/T base-pairs than controls (Phung et al., 1998; Rada et al., 1998;
Wiesendanger, 2000). Preferential mutations at G/C base pairs are also
seen in Mlh1/ mice (Ehrenstein and Neuberger, 1999; Schrader et al.,
1999) but are not as dramatic as those in Msh2/6 knockout mice, in-
dicating that Pms2 or Mlh1 are partially redundant. SHM is also com-
promised in mice with a ‘‘knockin’’ G674A mutation in the Msh2 gene
that inactivates the adenosine triphosphatase domain. This Msh2
mutation does not affect apoptosis signaling and allows mismatches to
be recognized, but it prevents Msh2 from initiating mismatch repair
(Martin et al., 2003). Thus, the effect of Msh2 on mutations at A/T base
pairs is not a reduced B-cell viability resulting from a general loss of
genomic stability.
The genetic data argue that a UG mismatch initially generated by
AID, or a GT mismatch caused by an error-prone polymerase during
BER, may sometimes be targeted for repair by the MMR pathway in B cells.
In this scenario, MMR would result in C ! T mutations on the transcribed
strand as well as on the nontranscribed strand (Fig. 3). Unlike the BER
pathway that removes U to cause a 1-base gap, the MMR pathway can
form a large gap on either side of the mismatch. If relatively large
MMR-generated gaps are equally likely to be formed on either DNA
strand, then AID-catalyzed C ! U deaminations at WRC hotspots may
occur with similar probabilities on both transcribed and nontranscribed
strands, leading to the absence of mutational strand bias, as well as
secondary mutations at WA sites nearby, using EP pols (Fig. 3).
Not surprising is the finding that MMR deficiencies can also have an
effect on CSR. MSH2-deficient mice display a two- to ten-fold reduction in
CSR (Ehrenstein and Neuberger, 1999; Schrader et al., 1999), and PMS2-
or MHL1-deficient mice exhibit two- to four-fold reduction in CSR
(Ehrenstein et al., 2001; Schrader et al., 1999). In addition, mice deficient
for exonuclease 1, an enzymatic contributor within the MMR pathway,
show impairments in both SHM and CSR similar to MSH2 mutant mice
(Bardwell, 2004). Although UNG-deficient mice show a strong defect
in CSR, neither UNG nor MMR deficiencies completely abolish CSR.
The overlapping activity of the BER and MMR repair pathways may
compensate for the remaining CSR function observed. The argument
that is now being made is that generic DNA repair mechanisms have
the potential to interject during CSR (Manis et al., 2002b) and SHM
(Neuberger et al., 2003), before completion of the immune-specific
pathways, and although it not fundamentally required, MMR, or perhaps
BER, appears to function in broadening the final mutational spectra
generated by the immune-specific processes.
G. Regulation of AID Expression in SHM and

Its Importance in Cancer Avoidance
Transcription of AID is highly regulated during normal B-cell develop-
ment. In humans and mice, its expression is restricted to centroblast cells
within germinal centers, and mRNA production in naı̈ve B-cells, cells that
have not yet encountered antigen, occurs predominantly within 48 hours
after in vitro stimulation (Greeve et al., 2003; Muramatsu et al., 1999).
Recent studies have identified at least three genes directly involved in
regulating AID expression: E2A (Sayegh et al., 2003), Pax5, and Id2
(Gonda et al., 2003). E2A and Pax5 are highly expressed in activated B
cells and are essential for their differentiation. E2A and Pax5 act by
binding to DNA at an E-box or Pax-box located in the regulatory region
of AID and activating its transcription (Gonda et al., 2003; Sayegh et al.,
2003). The Id2 gene encodes an inhibitor protein that negatively
regulates AID expression by interacting with E2A gene products, abolish-
ing their E-box binding activity. Examination of the putative regulatory
region of AID showed that the Pax5-binding site is absolutely required for
AID gene expression (Gonda et al., 2003).
Although antibody diversification benefits from AID deamination ac-
tion, its inappropriate expression can induce a significant increase in
genomic instability, which may lead to cancer. Not only are AID transcripts
and their splice variants readily detectable in B-cell non-Hodgkin
lymphomas (Greeve et al., 2003) and B-cell chronic lymphocytic leukemia
(Albesiano et al., 2003; McCarthy et al., 2003), but constitutive expression
of AID in transgenic mice also leads to the development of malignant T-
cell lymphoma, micro-adenomas, and adenocarcinomas in the lungs
(Okazaki et al., 2003). In addition, AID transgenic mice exhibit substantial
increases in point mutations, but not translocations, in expressed T cell
receptors and c-myc genes found in T cell lymphomas. Mutational distribu-
tion and specificity appear to be similar to those observed in B-cell
lymphoma (Pasqualucci et al., 2001) and AID-overexpressing cells in vitro
(Martin and Scharff, 2002; Martin et al., 2002; Yoshikawa et al., 2002),
indicating that AID-induced mutagenesis is responsible for tumorigenesis.
Similar to AID, ectopic expression of its homolog, Apobec-1, has been also
shown to promote liver cancer in transgenic mice (Yamanaka et al., 1995).
III. Apobec Protein Family

AID is homologous to the mRNA editing enzyme Apobec-1 (Table I),
sharing 34% amino acid identity (Muramatsu et al., 1999; Navaratnam et al.,
1993). Analyses of human genomic and EST data have revealed several
proteins with sequence homology to zinc-dependent deaminase domain
324 TIPPIN ET AL.
Table I
Human Apobec Protein Family of Nucleic Acid Deaminasesa
Gene Location Tissue expression Function
RNA deaminase
APOBEC-1 12p13.1 Small and large intestine mRNA apoB editing
DNA deaminase
AID 12p13 B lymphocytes SHM and CSR
APOBEC-3G 22q13.1 Spleen, breast, heart, thymus, Antiretroviruses
colon, stomach, kidney, uterus,
pancreas, placenta, prostate
Unknown
APOBEC-2 6p21 Cardiac and skeletal muscle Apobec-1 inhibitor?
APOBEC-3A 22q13.1 Keratinocytes ?
APOBEC-3B 22q13.1 Keratinocytes, colon ?
APOBEC-3C 22q13.1 Spleen, testes, heart, thymus, ?
Prostate, ovary, uterus
APOBEC-3D 22q13.1 Head and neck cancers ?
APOBEC-3E 22q13.1 ? Pseudogene
APOBEC-3F 22q13.1 B lymphocytes Antiretroviruses?
XP_092919 22q13.1 ? ?
XP_115170 12q23 ? ?
a
Adapted from Wedekind et al., 2003.
of Apobec-1 and AID (Anant et al., 2001; Jarmuz et al., 2002; Madsen et al.,
1999). A cluster of apobec-related proteins, Apobec3A to Apobec3G, and
an expressed gene (XM_092919) have been found on chromosome 22. In
addition, another gene (XP_115170), closely related to Apobec3G, is
located at position 12q23. These proteins have distinct expression profiles
and their functions remain largely unknown (Table I). Domain structures
of more than half of the members of the apobec family, such as Apobec-1
and AID, are characterized by the presence of the catalytic domain (CD)
and a pseudocatalytic domain (PCD) and separated by a linker region
(Fig. 4). However, other members like Apobec3 variants B, F, G, and its
mouse homolog CEM15, have CD-PCD-CD-PCD domain structure and
have probably evolved through gene duplication and divergence. On
the basis of biochemical properties, the members of this family can be
classified into two subclasses: RNA deaminases (APOBEC-1) and DNA
deaminases (AID and APOBEC3G) (Table I).
A. Apobec-1
Apobec-1, a founding member of this family, is responsible for C ! U
editing in apolipoproteinB mRNA at nucleotides 6666 and 6802, changing
Fig. 4. Domain structure of AID. AID is composed of a catalytic domain (CD)

containing a Zn+ binding consensus sequence and a pseudocatalytic domain (PCD),
separated by a linker region. The N-terminal region of AID is characterized by the
enrichment of positively charged lysine and arginine residues, possibly responsible for
its tight binding to nucleic acids. The C-terminal region of AID has been identified to
be essential for CSR. Amino acid changes identified in patients with impaired SHM and
CSR are indicated at the bottom.
a glutamine codon CAA to a stop codon UAA and a threonine codon to an

isoleucine, respectively (Teng et al., 1993). In addition, Apobec-1 may also
be responsible for mRNA editing of a tumor suppressor gene, neurofi-
bromin, in approximately a quarter of neurofibromatosis Type 1 patients
(Mukhopadhyay et al., 2002; Skuse et al., 1996). Site-specific C ! U
conversion by Apobec-1 requires its dimerization as well as an interaction
with at least one of two splice variants of ACF protein (ACF65 and ACF64).
The ACF proteins bind with high affinity to an AU-rich RNA known as the
mooring sequence located 30 of the edited cytidine and together with
Apobec-1 form a minimal editing complex (Dance et al., 2002; Mehta and
Driscoll, 2002). A number of other proteins have also been shown to
interact with Apobec-1 modulating its apoB mRNA editing activity in vitro,
but their involvement in vivo as auxiliary factors remains to be validated
(Wedekind et al., 2003). A homolog of Apobec-1, Apobec-2, can dimerize
with Apobec-1 and has been shown to inhibit its C ! U editing activity
(Anant et al., 2001; Liao et al., 1999). Although Apobec-1 is capable of
deaminating dC residues in vitro (Harris et al., 2003) and its overexpression
in E. coli induces C ! T transitions (Harris et al., 2002), there is no
evidence that Apobec-1 can deaminate DNA in human cells.
B. Antiretroviral Activity of Apobec3G, G ! A

Hypermutation in HIV and Other Retroviruses
One enigmatic feature of genetic variation of HIV and other retrovirus
genomes was G ! A hypermutation, characterized by high level of G ! A
base substitutions in the positive (mRNA) strand (Vartanian et al., 1991).
G ! A hypermutation can occur throughout the HIV genome with more
326 TIPPIN ET AL.
than a third of the total 2189 G residues being mutated. In some segments,
G ! A mutations are observed in up to 60% of G templates (Vartanian
et al., 2002). G ! A hypermutation is only seen during reverse transcrip-
tion, in which the retroviral RNA genome is copied into DNA in host cells.
A major clue to how G ! A hypermutation might happen came from the
discovery of the potent antiviral activity of an apobec protein family
member, Apobec3G, and a novel immune defense mechanism through
DNA dC deamination (Harris et al., 2003; Mangeat et al., 2003; Sheehy et al.,
2002; Zhang et al., 2003).
Similar to AID, Apobec3G was discovered in a substractive mRNA screen
for genes that specifically inhibit the replication of Vif-minus HIV virus
(HIV-1 strain) in nonpermissive cell lines (Sheehy et al., 2002). Vif, virus
infectivity factor, is a protein required by HIV-1 to replicate in certain non-
permissive cell lines, such as CEM15. It is now clear that Apobec3G targets
HIV by altering its genome through production of massive numbers of
G!A mutations (Harris et al., 2003; Mangeat et al., 2003; Zhang et al.,
2003). Apobec3G is incorporated into the Vif-minus HIV particles, and
upon their infection of naı̈ve T cells, the enzyme is released and works a C
deaminase on the first (minus) strand of DNA during reverse transcrip-
tion. As a consequene, about 1%–2% of all C residues in the minus strand
are deaminated to uracils. The mutations are preferentially observed at
third C residue in a 50 -YCC sequence.
Besides HIV, Apobec3G also acts on other retrovirus genomes such as
SIV, EIAV and MuLV (Harris et al., 2003; Mangeat et al., 2003). Recombi-
nant Apobec3G, purified from E. coli, has been shown to deaminate dC on
ssDNA in vitro (Harris et al., 2003). In addition, the enzyme, purified from
baculovirus-infected insect cells, exhibits a preferential specificity for CC
hotspots similar to what is seen in vivo, indicating that the enzyme itself is
solely responsible for the observed in vivo activity (P. Pham, R. Bransteitter
and M. F. Goodman, unpublished data). The presence of a massive
number of uracil residues in the minus strand of DNA could either lead
to its degradation by combined action of UNG and AP endonuclease, or to
the death of HIV virus by ‘‘error catastrophe,’’ whereby hypermutation in
essential genes no longer encodes functional viral proteins.
IV. Biochemical Perspective

The ability of higher organisms to mount an effective biodefense to
counter exposure to potentially life-threatening antigenic assault is central
to individual and species survival. By generating a diverse set of antibodies,
the immune response is generally capable of providing ample protection.
The conversion of low-affinity to high-affinity antibodies occurring by
somatic hypermutation and class-switch recombination allows antibodies

to carry out many effector functions and be distributed throughout the
body and secretions. Both processes are initiated by AID, an enzyme that
catalyzes transcription-dependent deamination of C residues. The recent
discoveries that the substrate for AID is single-stranded DNA and that AID
acting on ssDNA simulates SHM spectra, in the absence of additional
cofactors, will undoubtedly facilitate a deeper understanding into the mo-
lecular basis of CSR and SHM. This chapter has focused on the biochemical
role of AID during SHM, a ‘‘surface’’ that has just barely been ‘‘scratched.’’
There is a clear path ahead to address AID sequence specificity mechan-
isms, using relatively straightforward measurements of AID binding affi-
nities to ssDNA, dsDNA, and AID catalytic rates in C hotspot and coldspot
sequences. AID acts nondistributively on ssDNA, and there are methods
available for measuring enzyme processivity and scanning mechanisms in
‘‘simple’’ circumstances. However, all bets are off regarding the ease of
analyzing processivity, because AID is believed to be composed of a multi-
mer of identical subunits, and it is not known how many subunits can bind
to and act on DNA at any one time. AID has been shown to act in a
transcription-dependent manner using a model T7 RNA polymerase assay.
AID has also been found to deaminate C residues more aggressively using
a ‘‘transcription-like’’ bubble compared to ssDNA. The next step will be to
decipher how the mutational C-targeting AID is itself targeted to active
transcription complexes. Here it will be necessary to establish a eukaryotic
transcription system to investigate AID interactions with RNA polymerase
II, along with its numerous transcription factors. Somewhat further off is
the search for the SHM ‘‘Holy Grail’’—a stem-to-stern reconstitution of
SHM, incorporating MMR, BER and error-prone DNA polymerases acting
on chromatin.
Acknowledgments
This work was supported by grants from the National Institutes of Health, R37GM21422
and RO1ES012259. We thank Dr. Matthew D. Scharff for his patient and generous tutelage
and for reading and commenting on this chapter
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AUTHOR INDEX
A Amin, N. S., 155

Amin, S., 177, 210, 273
Aaltonen, L. A., 26 Anant, S., 311, 313, 323–325
Abagyan, R. A., 289 Andegeko, Y., 123
Abbondandolo, A., 21 Andersen, S., 25
Aboussekhra, A., 194 Anderson, C. W., 114
Abruzzo, L. V., 317, 323 Andersson, D. I., 241
Absalon, M. J., 7 Andersson, J., 312
Aburantani, H., 25 Angliker, H., 27
Ackerman, E. J., 21 Ansari, A., 53
Adachi, N., 267 Aono, N., 112
Adams, C., 151 Aoufouchi, S., 24, 147, 221, 320, 321
Adar, S., 194 Appella, E., 114, 119
Addona, T. A., 7, 9 Arad, G., 206, 249–252, 281
Adjiri, A., 194 Arai, T., 25
Agata, Y., 319, 323 Arakawa, H., 315
Aggarwal, A. K., 152, 212, 214, 233 Araki, K., 168, 185
Agustus, M., 168 Araki, M., 60, 61, 206, 207, 217
Ahern, H., 7, 9 Arany, Z., 23
Ahmad, M., 77, 90, 91, 93 Araujo, F., 94
Ahn, J. Y., 24, 113, 114 Aravind, L., 51, 267–269
Ahn, K., 53 Argos, P., 232
Akagi, J.-I., 180, 189, 220, 275 Ariyoshi, M., 9, 10
Aksenova, A. Y., 320 Ariza, R. R., 27
Albesiano, E., 323 Arlett, C. F., 207, 216
Ali, A., 26 Armout, C. D., 189
Ali, S., 28 Arnason, T., 286
Allan, J. M., 6, 25 Arndt, A., 291, 292
Allen, S. L., 323 Arnold, K. S., 323
Alley, J., 168 Arudchandran, A., 311
Allinson, S. L., 19, 21 Arunachalam, S. C., 326
Allis, D., 61 Arvai, A. S., 10, 15
Almouzni, G., 60 Asahara, H., 155, 156
Alon, U., 125, 127 Asano, T., 316
Alt, F. W., 308, 310–312, 316, Ashley, C., 297
318, 322 Asjo, B., 325
Altamirano, A., 19, 25 Ataujo, R., 91
Al-Tassan, N., 26 Augustine, M. L., 7
Altschul, S. F., 112 Avdievich, E., 121
Ames, B. N., 239 Avkin, S., 194
337
338 AUTHOR INDEX
Avril, M. F., 217, 219 Bassett, H., 16

Ayaki, H., 87 Basu, A. K., 19, 25
Ayyagari, R., 293 Batschauer, A., 93
Battista, J. R., 248
Batty, D., 53
B Baxter, S. M., 51
Bayer, P., 291, 292
Baarends, W. M., 297 Bayliss, J., 324
Baboshina, O. V., 286, 287 Baynton, K., 179
Bacchetti, S., 289 Beach, D., 113
Bachant, J. B., 299 Beard, B. C., 16
Bacher Reuven, N., 256 Beard, W. A., 19, 139, 143, 147, 208
Bachl, J., 311 Beardsley, D. I., 121
Bachmair, A., 287 Beaudenon, S., 291
Badie, C., 108, 116 Bebenek, K., 147–149, 151, 153, 184,
Baer, M., 82 208–212, 221, 237, 238, 269, 271, 320
Baer, R., 286 Becherel, O. J., 219, 242, 245, 249, 252
Bailly, V., 7, 185, 287, 288 Beck, A. K., 168
Bailone, A., 248 Becker, J., 291, 292
Baker, T., 150, 158 Beese, L. S., 256
Bakkenist, C. J., 111 Begley, T. J., 15
Balestra, M. E., 323 Belfort, M., 51
Baltimore, D., 118, 308 Belguise-Valladier, P., 248
Bambara, R. A., 173, 190 Bell, D. W., 114
Bandaru, V., 9 Bell, S. D., 247
Banerjee, A., 4, 10, 11, 13–15, 17 Bellacosa, A., 120, 121
Banerjee, S. K., 180 Bemark, M., 143, 183, 185
Banfalvi, G., 23 Bender, C. F., 122
Bankmann, M., 291 Benecke, A., 28
Bao, K. K., 13 Benjamin, D., 27
Barbanti-Brodano, G., 168 Benjamin, R. C., 21
Barbour, L., 280, 298 Benoist, C., 143, 308
Bardwell, P. D., 316, 322, 323 Berardini, M., 148
Barlow, C., 118 Bercovich, B., 289
Barlow, T., 10 Berdal, K. G., 17, 193
Barnes, D. E., 24, 25, 182, 297, 312, 316, 322 Berek, C., 143, 157, 310
Baroin-Tourancheau, A., 289 Beresford, P. J., 26
Barreto, V., 317 Beretta, B., 93, 94
Barrett, T. E., 10 Berg, P., 156
Barron, L. L., 317, 323 Bergoglio, V., 27
Bar-Sagi, D., 120, 122 Bergstein, M., 245
Barsky, D., 7 Berleth, E., 287
Bartek, J., 106, 107, 112–115, 122 Bermudez, V. P., 110
Bartel, B., 287 Bernad, A., 149
Bartholomew, B., 61 Bernards, A. S., 13
Bartkova, J., 112, 115 Bernier-Villamor, V., 292
Barton, J. K., 16 Bernstine, E. G., 168
Baskaran, R., 118, 121 Berrocal-Tito, G., 77, 91, 93
Basrur, V., 119 Bertocci, B., 143, 147, 157, 310
Bassett, E., 153 Bertram, J. G., 153, 251, 252
AUTHOR INDEX 339
Bertrand, Y., 308 Boorstein, R. J., 19, 25

Bertrand-Burggraf, E., 51, 52 Boosalis, M. S., 9, 208
Besmer, E., 308, 312, 316, 318 Bootsma, D., 53, 57, 94, 207, 216, 297
Bessho, T., 43, 55 Borden, A., 191
Best, J. M., 26 Borden, K. L., 285
Betz, U. A., 316 Bories, J. C., 143
Bezzubova, O., 185 Bork, P., 112
Bhagwat, A. S., 15, 312, 318, 320 Bosma, G., 312
Bhagwat, M., 7 Bosma, M., 312
Bhakat, K. K., 18, 23, 118 Botella, L. M., 248
Bharati, S., 10, 19 Botero, A., 23
Bharti, A., 118 Bottaro, A., 311
Bhattacharya, S., 323 Bouayadi, K., 27
Bichara, M., 248 Bouchier-Hayes, L., 115, 116
Bieser, G., 79 Boudsocq, F., 152, 153, 212–215, 217,
Bieth, A., 27 219, 233, 234, 236 –239, 244, 245
Bignell, C. R., 112 Boulares, H. A., 26
Bingham, C. M., 26 Bouly, J. P., 93
Bird, A., 14, 15, 25 Bowes Rickman, C., 94
Bisgaard, M. L., 26 Boyd, M. T., 155, 156
Bishop, S. M., 25 Brabec, V., 53
Biswas, T., 7 Bradbury, C. M., 23
Bjoras, M., 9 Bradley, A., 113, 122, 311
Black, E., 19 Braithwaite, D. K., 168
Blackburn, G. M., 17 Braman, J. C., 256
Bladl, A. R., 122 Bransteitter, R., 312, 316–320
Blanco, L., 147, 149 Branum, M. E., 48, 52, 55, 56
Blanco, M., 248 Brash, D. E., 87
Blandino, G., 118 Bregeon, D., 2
Blobel, G., 292 Brendel, M., 169
Bloom, L. B., 208, 221 Bresson, A., 179, 180, 191
Blumenstein, S., 245 Bresson-Roy, A., 179
Blunt, T., 308 Brick, P., 139
Boateng, M. A., 287 Bridges, B. A., 205
Bockrath, R., 56 Briggs, W. R., 93
Boddy, M. N., 285 Broekhof, J. L., 25
Bodepudi, V., 13 Bronson, R., 310
Bodmer, J. L., 122 Brooks, C. L., 115, 117
Boei, J. J. W. A., 185 Broomfield, S., 280, 281, 288, 289, 292,
Bogenhagen, D. F., 21, 152 293, 297–299
Bohr, V. A., 21, 44, 56, 145, 322 Brotcorne-Lannoye, A., 230, 240
Boiteux, S., 7, 18, 19, 24, 25, 193 Broughton, B. C., 207, 216, 217, 219
Boldogh, I., 7, 16 Brousse, N., 313, 317
Bolton, P. H., 7, 22–24, 55 Brown, J., 308
Bond, J. P., 9, 169, 170 Brown, K. D., 121
Bondo, B. E., 77, 91, 93 Brown, M., 289
Boner, W., 220 Brown, T., 10, 13
Bonner, C. A., 242 Broyde, S., 63
Boon, E. M., 16 Bruand, C., 139
Boone, C., 287 Bruck, I., 139, 150, 206, 245, 249, 281
340 AUTHOR INDEX
Brudler, R., 75, 77, 91 Carrozzino, F., 21

Bruner, S. D., 7, 9, 11, 12, 15, 17 Carson, M., 213, 245
Brusky, J., 289 Carswell-Crumpton, C., 281
Bryans, M., 310 Cartegni, L., 325
Bucher, P., 112 Carter, D. M., 207
Buerstedde, J.-M., 185, 315 Carter, K. C., 168
Bugg, C. E., 286 Carter, T., 308
Buijs, R., 94 Casali, P., 183, 321
Bulavin, D. V., 119 Caseira-Cabral, R. E., 194
Bullock, H. A., 26 Casellas, R., 308, 312
Buluwela, L., 28 Cashmore, A. R., 73, 75, 77, 90–92, 94
Bunn, H. F., 23 Caspari, T., 110
Bunny, K. L., 241 Casselman, R., 110
Bunting, K. A., 245, 247, 252 Cassier, C., 170
Burant, C. F., 325 Cassier-Chauvat, C., 168, 194
Burckhardt, S. E., 248 Castano, I. B., 151
Burgers, P. M., 22, 110, 175, 191, 192, Castle, B. E., 317, 323
293, 294 Catalan, N., 313, 316, 317
Burgess, S. M., 47 Cavazzana-Calvo, M., 308
Burma, S., 111 Cazaux, C., 27
Burnouf, D. Y., 243, 245, 246 Celis, J. E., 324
Burrows, C. J., 6 Ceriani, M. F., 93
Bustin, M., 308 Cerosaletti, K. M., 113
Buterin, T., 53, 63 Cerritelli, S., 311
Cerrutti, M., 321
Cerutti, A., 183
C Cesare, A. J., 110
Cevasco, M., 21
Cabral-Neto, J. B., 194 Chabes, A., 152
Cadet, J., 211 Chae, S.-K., 168, 171
Cado, D., 121 Chaganti, R. S., 323
Calandrini, D. M., 183, 194, 315 Chambon, P., 28
Caldecott, K. W., 21, 24, 145, 147 Chan, B. H., 325
Callebaut, I., 308 Chan, E., 18
Campbell, J. L., 151, 155 Chan, L., 325
Canagarajah, B., 59 Chan, M. K., 19, 25
Canitrot, Y., 27 Chan, T. A., 107
Canman, C. E., 113, 114, 118 Chanet, R., 170, 194
Cann, I. K., 139 Chaney, S. G., 153, 210
Cao, C., 14 Chang, D. Y., 6, 16
Cao, X., 27 Chang, G. J., 121
Cappelli, E., 21 Chapados, B. R., 21, 22
Cappione, A. J., 325 Chapuis, J., 139
Carattini-Rivera, S., 113 Charron, J., 308
Carlson, C., 311 Chaskara, V., 122
Caron, G., 326 Chau, B. N., 116, 118
Carpenter, A. J., 108, 116 Chau, V., 285, 287
Carpenter, P. B., 112 Chaudhuri, J., 310–312, 316, 318
Carr, A. M., 110 Chaung, W., 25
Carrodeguas, J. A., 152 Chauveau, C., 311
AUTHOR INDEX 341
Cheadle, J. P., 26 Clarkin, K., 108

Cheah, H., 311 Clarkson, S. G., 22–24
Cheh, A. M., 210, 211, 273 Cleaver, J. E., 44, 60, 207
Chen, B. J., 9 Cocea, L., 147
Chen, B. P., 111 Cogne, M., 311
Chen, C.-C. A., 268 Cohen, Y., 24
Chen, D., 28 Coin, F., 248
Chen, D. J., 111 Cole, P. A., 118
Chen, D. S., 18 Coleman, J. E., 46
Chen, J., 112, 311 Collado, P., 248
Chen, P., 114 Collins, J. E., 324
Chen, T. T., 108, 116 Concannon, P., 113
Chen, X., 143, 158, 311 Cong, F., 119, 122
Cheng, H., 310 Connaughton, J. F., 212, 230, 232
Cheng, L. S., 117 Connolly, J. A., 220
Cheng, X., 15, 81, 88 Constantinou, A., 22–24
Cheng, Y. C., 19 Cook, W. J., 285, 286
Chernov, M. V., 189 Coombes, K. R., 317, 323
Chester, A., 324 Coombes, R. C., 28
Cheung, A., 114 Cooper, E., 325
Cheung, K.-J., 189 Copeland, W. C., 139, 148, 152, 157
Chiapperino, D., 210, 211, 273 Coquerelle, T., 27
Chini, C. C., 112 Cordon-Cardo, C., 117
Chiolo, I., 298 Cordonnier, A. M., 217, 219, 242, 249
Chiorazzi, N., 323 Corneo, B., 308
Chipuk, J. E., 115, 116 Cortes, P., 308
Chittenden, T., 115, 116 Cortez, D., 110, 111, 113
Chmiel, N. H., 13, 16, 26 Costanzo, A., 117, 118
Choi, B. S., 211 Cotticelli, M., 312
Choi, J. D., 326 Coull, B., 216, 217
Choi, Y., 27 Courcelle, J., 248, 281
Chou, K. M., 19 Cox, L. S., 21
Chow, B. L., 168, 289, 293, 297 Cox, M. M., 255, 281
Chow, K. H., 281 Crabtree, M., 26
Christen, R. D., 118 Craig, H. M., 325, 326
Christensen, R. B., 168–170, 185, 187, 188 Craig, L., 122
Christman, M. F., 151 Cranston, A., 322
Chu, G., 64, 119, 122 Creighton, S., 208, 221
Chua, K., 311, 312, 316, 318 Crick, F. H. C., 158
Chua, N. H., 93 Critchlow, S. E., 149
Chumakov, P. M., 189 Croce, C. M., 155, 168, 187, 189, 275
Chung, U., 18 Cromwell, C. C., 317, 323
Chyan, J. Y., 19 Cross, T. G., 122
Ciechanover, A., 283, 289 Crouch, R., 311
Citaterna, M. H., 94, 96 Crowley, D., 117
Clark, A. B., 121 Cui, X. S., 113
Clark, D. R., 171 Cullis, P. M., 6
Clark, S. C., 325 Cumano, A., 143, 183, 185
Clarke, A. R., 25 Cunningham, M. L., 26
Clarke, N. D., 13 Cunningham, R. P., 7, 9, 15, 19, 20, 25
342 AUTHOR INDEX
Cuomo, C., 308 Delbos, F., 147

Curran, T., 18, 23, 26 de Lera, L. T., 147
Curren, R. D., 220 del Mazo, J., 147
Delsol, G., 27
DeLucia, A. M., 233
D DeMayo, F., 113
Demengeot, J., 308
Dahan, A., 143, 147 Demple, B., 18, 192, 193
Dahlen, M., 110 de Murcia, G., 23, 24, 316
Daiyasu, H., 75, 77, 91 Deng, Y., 114
Dalla-Favera, R., 323 Deng, Z., 9
Dalton, T. P., 265 Denissenko, M. F., 265
Daly, G., 25 Derbyshire, V., 51
Damle, R. N., 323 de Ruiter, P. E., 77, 79
D’Amours, D., 112 Dervyn, E., 139
Dance, G. S., 325 De Smet, A., 143, 157, 310
Daniel, R., 139 Desnoyers, S., 26
Dansereau, J. T., 51 Dessing, M., 311
Dantzer, F., 24 Desterro, J. M., 292
Dargemont, C., 292 Deutsch, W. A., 23
Darlington, T. K., 93 de Villartay, J., 308
Das, G., 168, 169 Devoret, R., 248
Das, S., 311 de Weerd-Kastelein, E. A., 207, 216
Datta, A., 155, 170 de Wind, N., 185, 316
Datta, M., 308 de Wit, J., 25, 57, 94, 297
Daune, M., 23 Dianov, G. L., 19, 21, 25, 145
Davey, S., 110, 113 Dianova, I. I., 19, 21
David, L., 27 Diaz, M., 182, 183, 321
David, S. S., 13, 16, 26 Dickerson, S. K., 312, 316, 318
Davidson, L., 308, 310–312 Dierich, A., 143, 308
Davidson, N. O., 311, 313, 323–325 Dighiero, G., 317
Davies, D. R., 26 Dikkes, P., 310
Davies, S. L., 143, 183, 185 Di Leonardo, A., 108
Davis, A., 311 Ding, C. K., 26
Davis, H. L., 113, 114 Dinner, A. R., 17
Davis, R. J., 119, 120, 122 Di Noia, J., 157, 182, 316, 322
Dawut, L., 91, 94 Dionne, I., 247
Deacon, E., 122 Dittmar, G., 286
Dean, K., 287 DiTullio, R. A., Jr., 112
de Boer, J., 316 Djamei, A., 93
de Chasseval, R., 308 Doddridge, Z. A., 2
Decourt, C., 311 Dodson, L. A., 248
Defais, M., 280 Dodson, M. L., 7, 9
Deisenhefer, J., 77, 78, 81 Doetsch, P. W., 2, 22–24
de Jong, A. W. M., 168, 171 Dogliotti, E., 21
Delagoutte, E., 51, 52 Doherty, A. J., 247
de La Rubia, G., 24 Dohmae, N., 206, 207, 217
Delarue, M., 232 Dohmen, R. J., 287
De Las Penas, A., 151 Dolwani, S., 26
De Laurenzi, V., 117 Dominguez, O., 147, 149
AUTHOR INDEX 343
Donker, I., 25 Eisenberg, W., 194

Doorly, M., 119 Eisler, H., 170
Dornan, E. S, 220 Eker, A. P. M., 53, 77, 79, 94
Dosch, J., 27 Elder, R. H., 25, 26
Dover, J., 287 Elenitoba-Johnson, K. S., 317, 323
Downey, K. M., 173, 190 Elia, A. J., 114
Drapkin, R., 53, 59, 122 Elias, S., 283
Driscoll, D. M., 325 Elledge, S. J., 104, 110–114, 155, 299
Drohat, A. C., 14, 17 Ellenberger, T., 6, 9–11, 14, 17, 45, 152,
Droin, N. M., 115, 116 194, 212–214, 233
Dua, R., 151, 155 Ellis, A., 26
Dudkin, E. A., 93 Ellison, M. J., 284–286, 289, 290, 297
Dudley, D., 311, 318, 322 Elowitz, M. B., 125, 127
Due, M., 313 Emery, P., 93, 94
Dufourcq-Labelouse, R., 313, 317 Emmerson, P., 26
Dumanski, J. P., 324 Engels, K., 26
Dumas, G., 317 Engelward, B. P., 9, 25, 316
Dumas, P., 243, 245, 246 Epe, B., 25
Dunand, J., 51 Epstein, J. A., 207, 216
Duncan, B. K., 14 Erez, N., 24
Duncan, G., 114 Eritja, R., 206, 242, 249, 281
Dunham, I., 324 Errington, J., 139
Dunnick, W., 122 Ersoy, F., 313, 317
Dunstan, H. M., 168 Esashi, F., 112
Duong, J. K., 112, 114 Escalante, C. R., 152, 212, 214, 233
Durandy, A., 313, 316, 317 Escobar, S. R., 118
Durocher, D., 112, 114 ESCODD, 15
D’Urso, G., 155 Esposito, G., 143, 183, 185, 271, 316
Dutreix, M., 248 Essers, J., 316
Estivill, X., 289
Etienne, H., 241–243
E Eto, T., 323
Evan, G. I., 285
Earnshaw, W. C., 26 Evans, D. G., 26
Ebbert, A., 56 Evans, D. H., 75
Ebisu, S., 18 Evans, E., 53, 55
Ebright, J. N., 94 Evans, J., 308
Eccles, D., 26 Evans, R. M., 28
Echols, H., 242, 248, 249
Ecker, D. J., 287
Edelmann, W., 316, 322 F
Edwards, R. A., 284, 285, 290
Eeken, J. C. J., 168, 171, 185–187 Fabre, F., 194
Egly, J.-M., 53, 55 Fagarasan, S., 182, 311, 313
Ehrenstein, M. R., 316, 322 Faili, A., 147, 221, 320, 321
Ehrlich, S. D., 139 Fajnok, M. F., 183
Eichman, B. F., 14, 17 Falck, J., 106, 107, 112, 114, 115, 122
Einhorn, L. H., 26 Falcovitz, A., 23
Eisen, J. A., 63 Fan, J., 323
Eisenberg, M., 13 Fan, M., 323
344 AUTHOR INDEX
Fan, Z., 26 Frank, K., 310

Fanciulli, M., 118 Franklin, K. A., 52, 81
Fanklin, W. A., 87 Fraumeni, J. F., 114
Fath, D., 312 Frayling, I., 26
Favaudon, V., 118 Frechet, M., 27
Fawcett, H., 217, 219 Freemont, P. S., 285
Fay, D. S., 112, 114 Freidberg, E. C., 141
Feaver, W. J., 269, 271, 274 Fridman, J. S., 108, 115
Fernandes, A. S., 14 Fried, L., 308
Fernández de Henestrosa, A. R., 149, Friedberg, E. C., 57, 59, 61, 102, 143, 147,
216, 217 152, 155, 168, 189, 205–207, 220, 229,
Ferrell, L. D., 323 265, 267–269, 271, 274, 275
Ferrini, R., 308, 310, 312 Friedli, M., 326
Feunteun, J., 112 Friend, S. H., 168
Fiala, K. A., 234, 236, 237 Fritz, G., 19, 23, 26
Fidalgo, P., 26 Fromme, J. C., 4, 9–15, 17
Fink, D., 118 Frosina, G., 21
Finkel, S. E., 206, 240 Fruechte, E. M., 94, 96
Finley, D., 283, 286, 287 Fuchs, R. P., 51, 52, 152, 153, 155, 168, 179,
Finnie, N., 308 180, 191, 206, 217, 219, 229, 232–235,
Fischer, A., 308, 313, 316, 317 237, 239–243, 245, 246, 248–252, 256
Fischer, R. L., 27 Fugmann, S., 310
Fischhaber, P. L., 152, 155, 189, 220, 269, Fujii, S., 234, 235, 242, 243, 245, 246,
271, 274, 275 250–252, 256
Fishel, R., 155, 168, 187, 189, 275, 322 Fujii-Kuriyama, Y., 266
Fisher, C. L., 7 Fujimoto, H., 266
Fisk, H. A., 286 Fujimoto, K., 23
Flaherty, D. M., 23 Fujiwara, Y., 310
Flajnik, M. F., 183, 315, 321 Fukao, T., 316
Flatter, E., 24, 143, 147, 221, 321 Fukasawa, M., 267
Flavell, R. A., 120, 122 Fukita, Y., 316
Fleming, N., 26 Fukuda, M., 25
Flores, E. R., 117 Fukuda, R., 230, 239
Fluke, D. J., 79 Fukuda, S., 168, 173
Foiani, M., 155, 298 Fulco, M., 118
Fontanie, T., 168, 289 Funahashi, T., 323
Fontemaggi, G., 118 Furuichi, M., 25
Foote, R. S., 6 Fuss, J., 151, 155, 156
Foray, N., 118 Futcher, B., 292
Fornace, A. J., Jr., 119 Fygenson, K. D., 208
Fortini, P., 21
Forveille, M., 313, 316, 317
Foster, P. L., 148, 206, 240 G
Foster, R. S., 26
Fox, M., 289 Gaarde, W. A., 119
Francis, A. W., 13, 16 Gaddis, N. C., 326
Francis, L., 241 Gaiddon, C., 23
Frank, E. G., 143, 147, 148, 177, 184, Galan, J. M., 286
194, 206–212, 216, 221, 249, 250, Galante, J., 108, 116
252, 281, 321 Gale, J. M., 59, 60
AUTHOR INDEX 345
Galgoczy, D. J., 108, 109 Gilboa, R., 14

Gali, R. R., 286 Gilfillan, S., 143, 308
Galibert, F., 194, 248 Gill, D. M., 21
Galkin, A. P., 320 Gillespie, D., 19
Gallant, M., 26 Giovani, B., 93
Gallinari, P., 19 Girard, P. M., 7, 114
Gamper, H., 43, 51 Gisbourne, J., 324
Ganesan, A. K., 7 Gius, D., 23
Gao, L., 326 Glassner, B. J., 9–11, 27, 170, 193
Gao, T., 267, 274 Glastre, C., 316, 317
Gao, Y., 310 Glickman, B. W., 312
Garcia-Diaz, M., 147, 149 Glover, B. P., 150
Garcia-Ortiz, M. J., 147 Glover, J. N., 284, 285, 290
Garcia-Palomero, E., 147 Godin, I., 183, 185
Gareau, Y., 26 Godindagger, I., 143
Gares, M., 27 Godoy, V. G., 250
Garkavtsev, I., 189 Goff, S. P., 119, 122
Garritsen, V. H., 217, 219 Gogos, A., 13
Gary, R., 21 Golan, G., 14
Gary, S. L., 293 Gold, B., 25
Gasparutto, D., 211 Gold, L. S., 239
Gasser, V., 250–252 Goldberg, M., 112, 114
Gatter, K. C., 26 Goldberg, R. B., 27
Gaudreau, A., 23 Goldfinger, N., 23
Gauger, M., 153 Golding, G. B., 312
Geacintov, N. E., 63, 153, 173, 177, 178, 210, Goldsby, R. E., 143, 158
241, 242, 273 Goldstein, G., 283
Gearhart, P. J., 143, 183–185, 208, 212, 216, Golinelli, M. P., 13
221, 312, 316, 320–322 Golshani, A., 287
Gehring, M., 27 Gonda, D. K., 287
Geissmann, F., 313, 317 Gonda, H., 319, 323
Gellert, M., 157, 308 Gong, J. G., 117, 118
Gennery, A., 313, 317 Gong, Z., 27
Gentil, A., 194 Gonzalez, F. J., 265
Georgescu, R. E., 243 Gonzalez, M., 250
Gerchamn, S. E., 14 Gonzalez, M. A., 149
Gerlach, V. L., 143, 267–269, 271, 274 Gonzalez, S., 117
Gerlt, J. A., 7 Gonzalez-Barrera, S., 149
Getzoff, E. D., 75, 77, 91 Goodman, M. F., 137, 139, 150, 152,
Geva-Zatorsky, N., 125, 127 153, 156, 157, 168, 172, 206, 208,
Ghavidel, A., 23 209, 212–214, 221, 229, 233, 235–237,
Ghirlando, R., 122 239–243, 249–252, 255, 256, 273, 281,
Giaccia, A. J., 115, 117 312, 316–320
Giannoni, F., 323 Goosen, N., 51
Gibbs, P. E. M., 167, 168, 171–176, 179, 180, Goossens, T., 323
187, 188, 190, 191 Gorka, C., 308
Gibson, N. J., 13 Goswami, P. C., 23
Giedroc, D. P., 46 Gottesman, M. E., 57
Giercksky, K. E., 6 Gottlieb, T., 308
Gietz, R. D., 170 Gotway, G., 267–269
346 AUTHOR INDEX
Grallert, B., 155 Halazonetis, T. D., 112

Granek, J. A., 13 Hall, J. C., 93, 94
Grawunder, U., 310 Hamilton, J. W., 43
Gray, C., 216, 217 Hamlin, R., 139
Gray-Schopfer, V., 311 Hamm-Alvarez, S., 77
Green, D. R., 115, 116 Han, D.-M., 168, 171
Greenberg, M., 310 Han, K.-Y., 168, 171
Greenblatt, J. F., 287 Han, S., 21, 22
Greeve, J., 317, 323 Hanada, K., 267
Griffin, E. A., Jr., 93 Hanaoka, F., 53, 60, 61, 141, 143, 152, 153,
Griffin, P. R., 26 168, 177, 180, 189, 206–212, 215–217,
Griffith, J. D., 51, 81, 110, 111 219–221, 229, 233, 239, 269, 273–275,
Grigorian, I. A., 189 320, 321
Grindley, N. D., 233, 237 Hanawalt, P. C., 44, 45, 52, 56, 57, 63,
Groisman, R., 122 64, 248, 281
Grollman, A. P., 6, 7, 9, 13, 14, 239, 273 Hang, B., 25
Gromov, P., 324 Hanna, M., 289, 293
Grootegoed, J. A., 297 Hannon, M., 27
Grossman, L., 7 Hanson, I. M., 285
Gruz, P., 206, 232–235, 237, 239–242 Hara, R., 59, 61, 64
Gu, H., 143, 316 Haracska, L., 155, 172–178, 190–192,
Gu, J., 122 195, 206, 208–211, 215, 219, 243,
Gu, W., 115, 117 272, 274
Gu, Y., 6, 16, 19, 308, 312 Harada, J. J., 27
Guan, Y., 19, 20 Harada, N., 114
Gudkov, A. V., 189 Harashima, H., 233, 239
Gueranger, Q., 221, 321 Hardeland, U., 15, 28
Guerrera, S., 155 Hardigree, A. A., 168, 186, 206, 230
Guibourt, N., 7 Hardwich, K. G., 155
Guillet, M., 18, 193 Hardy, R., 310
Guntuku, S., 113 Harfe, B. D., 170
Gunz, D., 22, 23, 24 Harriman, G., 311
Guo, C., 143, 155, 189, 220, 271, 275 Harris, A. L., 26, 152, 155
Guo, D., 153, 175–177, 190, 210, 273 Harris, C. C., 24
Gupta, M., 18 Harris, R. S., 157, 315, 322, 325, 326
Gupta, N., 147 Hart, S. M., 28
Guthrie, C., 47 Hartwell, L. H., 108, 109, 168, 189
Guy, J., 25 Harvey, C., 289
Hase, Y., 168, 171
Hasegawa, R., 9, 25
H Haseltine, W. A., 87
Hastings, P. J., 170, 206, 241
Haas, A. L., 286, 287 Hata, Y., 174, 180, 191, 194
Haas, B. J., 19, 20 Hatada, E., 230, 239
Haber, J. E., 156 Hatahet, Z., 274
Haguenauer-Tsapis, R., 286 Hauschild, J., 315
Hahn, S., 61 Haushalter, K. A., 10, 25
Hahn, W. C., 117 Havener, J. M., 153
Haines, D. S., 155, 156 Hay, R. T., 292
Haire, L. F., 112, 114 Hayakawa, H., 150
AUTHOR INDEX 347
Hayakawa, K., 310 Hingorani, M., 243

Hayakawa, S., 116 Hinkle, D. C., 152, 167, 168, 171–177,
Hayashi, I., 234, 235, 239 179, 186, 188–191, 205, 206, 229, 232
Hayashi, K., 239 Hiom, K., 308
Hays, L. E., 143, 158 Hippou, Y., 25
Hazel, J. C., 206 Hirano, M., 25
Hazra, T. K., 9, 18, 23 Hirao, A., 114
He, M., 112 Hirata, Y., 79, 84, 85
Hearst, J. E., 43, 47, 51, 52 Hirota, K., 23
Heelis, P. F., 79, 83–85 Hitomi, K., 75, 77, 88–89, 91, 94, 96
Hegde, V., 23 Hladik, C. L., 143, 267, 271, 274
Heidenreich, E., 170 Hobeck, S.L., 170, 184
Heidorn, K., 317, 323 Hochstrasser, M., 283, 285, 291
Heidt, J., 287 Hodges, A. K., 26
Heinimann, K., 26 Hodgson, S. V., 26
Heller, H., 283 Hoege, C., 155, 187, 219, 243, 274, 288,
Helquist, S. A., 215 292–294, 297, 298, 300
Hemmingsen, S. M., 289 Hoeijmakers, J. H., 9, 25, 53, 57, 297
Henderson, B., 122 Hoekstra, M. F., 110–112
Henderson, J. O., 324, 325 Hoffmann, J. S., 27
Henderson, S. T., 291 Hoffmeyer, M. R., 122
Hendrich, B., 15, 25 Hofmann, K., 110, 112
Hendry, J. H., 108 Hofmann, R. M., 286, 289, 290
Henning, K. A., 57 Hofseth, L. J., 24
Henriques, J. A. P., 169, 170 Hokijmakers, J. H. J., 94
Henriquez, N. V., 122 Holbrook, S. R., 43, 81–83
Henry, M., 326 Hollis, T., 14
Hensen, E. S., 192, 193 Holmes, A. M., 156
Herman, T., 18 Holmes, E. W., 26
Hermeking, H., 107 Holpert, M., 51
Hermine, O., 316, 317 Holzmann, V., 170
Herrera, G., 248 Honda, K., 116
Herrup, K., 308 Hong, S. H., 325
Hershko, A., 283 Honjo, T., 182, 311, 313, 315, 317, 323
Hess, D., 27 Hoogerbrugge, J. W., 297
Hess, P., 120, 122 Hopfield, J. J., 47
Hessels, J. K. C., 77 Hopfner, K. P., 122
Heude, M., 194 Horiuchi, T., 18
Hey, T., 53 Horton, J. K., 21
Hiai, H., 323 Hosaka, Y., 217, 219
Hickson, I. D., 19, 24, 26 Hosfield, D. J., 7, 19–22, 110
Hidaka, M., 18 Hoss, M., 22–24
Hideki, K., 266 Hostomsky, Z., 24
Higashimoto, Y., 119 Hou, E., 208
Higley, M., 16 Howard-Flanders, P., 280
Higuchi, K., 18 Howell, S. B., 118
Higuchi, Y., 77, 79 Howley, P. M., 291
Hikida, M., 266 Hrivnak, G., 25
Hill, C. P., 285, 288 Hryciw, T., 280, 281, 288, 289, 292, 299
Hindges, R., 53 Hsiao, J., 112, 114
348 AUTHOR INDEX
Hsieh, C. L., 22 Irwin, M. S., 117

Hsieh, M. M., 23 Isaacs, R. J., 63
Hsu, D. S., 51, 53, 55, 77, 81, 87–91, 94 Ishibashi, T., 150
Huala, E., 93 Ishii, C., 168, 171, 186
Huang, J.-C., 43, 53, 55 Ishii, H., 155, 168, 187, 189, 275
Huang, L., 291 Ishikawa, T., 88, 94, 96, 212, 266
Huang, L. E., 23 Ishiko, T., 119
Huang, M.-E., 194 Ishino, Y., 139
Huang, S. J., 11, 13–15 Ishiura, M., 75, 77, 91
Huang, Y., 119 Isselbacher, K. J., 114
Hubscher, U., 21, 53, 137, 156 Ito, J., 168
Huibregtse, J. M., 285, 291 Ito, S., 317
Huletsky, A., 23 Ito, T., 61
Hunninghake, G. W., 23 Itoh, M., 25
Hunter, T., 118 Itoh, S., 274
Hunter, W. N., 13 Itoh, T., 64, 121, 217, 219
Hurwitz, J., 21, 110, 195, 219, 274, 293 Itzhaki, J. E., 108, 116
Husain, I., 77, 81–83 Iwai, S., 9, 10, 18, 53, 61, 87–89, 141, 153,
Hussain, S. P., 24 174, 180, 182, 191, 206–208, 210–212,
Hutchinson, F., 237 215–217, 221, 239, 269
Hwang, B. J., 119, 122 Iwasaki, H., 248, 281
Hwang, G. S., 211 Iyer, N., 57, 59
Hwang, J. R., 53, 55 Izumi, T., 7, 9, 18–20, 23, 192
Hwang, W. W., 287
J
I
Jack, M. T., 114
Iacomini, J., 308 Jacks, T., 117
Ianculescu, A. G., 287 Jackson, S. P., 110, 112, 114, 149, 247
Ichihara, M., 189, 275 Jacobs, H., 316
Ichikawa, Y., 14, 17 Jacobs, M. A., 143, 183, 185
Ichinose, M., 266 Jacobsen, S. E., 27
Ide, H., 9, 25 Jaenicke, R., 291, 292
Iden, C. R., 7, 9 Jager, J., 139
Igarashi, T., 18 Jaiswal, A., 23
Iglesias-Ussel, M., 322 Janawalt, P. C., 44, 56
Ihara, M., 77, 87 Janda, J., 317, 323
Ikeda, S., 7 Janel-Bintz, R., 239, 241, 242
Ikegawa, M., 317 Jankovic, M., 318, 320
Ikenaga, M., 87, 89 Janniere, L., 139
Imai, K., 116, 316, 317 Jarillo, J. A., 91
Impellizzeri, K. J., 293 Jarima, N., 87, 89
Inaka, K., 77, 79 Jarmuz, A., 324
Inman, R. B., 281 Jaspers, N. G., 217, 219
Innerarity, T. L., 323 Jaspers-Dekker, I., 297
Inoue, H., 168, 171, 186, 297 Jayaraman, L., 23
Inoue, S., 118 Jeffrey, L. C., 285
Inoue, Y., 77, 79 Jeffrey, P. D., 285, 291
Inui, T., 87 Jeggo, P., 118, 308, 310
AUTHOR INDEX 349
Jensen, D. E., 46 Kahyo, T., 292

Jensen, E. L., 168 Kai, M., 110, 243, 275
Jentsch, S., 155, 187, 219, 243, 274, 283, 285, Kaina, B., 19, 23, 26, 27
286, 288, 291, 292–294, 297–300 Kajiwara, K., 186
Jerina, D. M., 153, 177, 210, 211, 239, Kakazu, N., 323
241, 273 Kaklamanis, L., 26
Jeruzalmi, D., 243 Kakolyris, S., 26
Jessberger, R., 156 Kakutani, T., 27
Jeusset, J., 194 Kamath, R., 121
Jiang, Y. L., 2, 6, 14, 16, 17 Kamide, R., 121, 217, 219
Jin, B., 121 Kamiya, H., 233, 239
Jin, S., 118 Kamiya, K., 155, 168, 173, 189, 220, 275
Jinks-Robertson, S., 170 Kaneda, Y., 61
Jiricny, J., 10, 15, 19, 28 Kanehisa, M., 75, 77, 91
Joachimiak, A., 293 Kaneko, H., 316
Joazeiro, C. A., 285 Kaneko, M., 93, 94
Johansen, R. F., 17, 193 Kang, C., 81, 82
Johansson, E., 175, 191, 192 Kang, D. H., 114
Johnson, A. W., 18 Kanjo, N., 180, 189, 220, 275
Johnson, E. S., 292 Kanke, Y., 233, 239
Johnson, F., 13 Kanno, S., 9, 25, 94
Johnson, J. L., 77 Kannouche, P. L., 216, 217, 220, 243
Johnson, K. A., 234 Kanter-Smoler, G., 110
Johnson, L., 27 Kanuri, M., 210
Johnson, P., 24 Karcher, A., 122
Johnson, R., 308 Karin, M., 119, 286
Johnson, R. E., 18, 152, 172, 173, 175–177, Karnitz, L. M., 110
190–192, 195, 206–212, 214, 215, 217, Karplus, M., 17
219, 233, 243, 271–274 Karran, P., 145
Johnson, S. J., 256 Kasahara, K., 316
Johnston, M., 287 Kasahara, Y., 316
Jones, A. M., 322 Kashiwagi, T., 9, 10
Jones, S., 26 Kasmer, C., 184, 221, 320
Jones, S. N., 120, 122 Kaspárková, J., 53
Jonson, R. E., 291 Kassabov, S. R., 61
Jonsson, Z. O., 21 Kastan, M. B., 111, 112, 115, 117, 118
Jordan, S., 26 Katakai, T., 319, 323
Jorns, M. S., 79, 83 Katayama, T., 18
Jost, J. P., 27 Kato, T., 206, 248, 267, 281
Jost, Y. C., 27 Kato, T., Jr., 267
Joyce, C. M., 139, 233, 237 Katori, N., 217, 219
Juarez, R., 147, 149 Kaufmann, S. H., 26
Jung, S., 311 Kavakli, I. H., 77, 91, 93
Kavli, B., 10, 316, 317
Kawaguchi, N., 174, 180, 191
K Kawamura, K., 266, 267
Kawano, M., 267
Kaelin, W. G., Jr., 117, 118 Kawashima, A., 53, 121
Kafer, E., 171 Kawate, T., 7, 9
Kaguni, L. S., 152 Kay, S. A., 93, 94
350 AUTHOR INDEX
Kaylor, L., 311, 318, 322 Kitao, H., 119

Kayserili, H., 313, 317 Klapacz, J., 312, 318, 320
Kazantsev, A., 55, 77, 90, 91, 94 Klapper, W., 317, 323
Keating, K. M., 46 Kleijer, W. J., 217, 219
Keating, M. J., 317, 323 Klein, H., 194
Kedar, P., 21 Klein, U., 143, 183, 185, 316
Keegan, K., 112 Klinman, N. R., 183, 321
Keeney, S., 121, 299 Klungland, A., 21–25, 297
Kelley, M. R., 23, 26 Kneitz, B., 322
Kelly, R. C., 46 Kobayashi, K., 9, 25, 94, 174, 180, 191
Kelly, U., 94 Kobayashi, S., 235, 236, 237, 239
Kelly, V. P., 25 Koffel-Schwartz, N., 248
Kelman, Z., 150, 293 Kofoid, E., 241
Kennedy, S., 324, 325 Kogoma, T., 281
Kenny, M. K., 21 Kohler, G., 308
Kenter, A., 308, 312 Kohlhagen, G., 18
Kenyon, C. J., 230 Kohn, K. W., 18
Keranen, S., 151 Koken, M. H., 297
Kesti, T., 151 Kokoska, R. J., 141, 153, 210, 211, 214,
Khamlichi, A. A., 143, 183, 185, 311 233, 237, 238
Kharbanda, S., 118, 119 Kolbanovskiy, A., 241, 242, 273
Khoung, C., 311, 312, 316, 318 Kolchanov, N. A., 312, 318
Kiefer, J. R., 256 Kolodner, R. D., 121, 155
Kiener, A., 77, 82 Komori, A., 183, 321
Kikuchi, H., 116 Komori, H., 77, 79
Kikuchi, Y., 292 Komori, T., 308
Kilbey, B. J., 180 Komoro, K., 25
Kim, J., 312 Kondo, K., 117
Kim, J. K., 211 Kondo, N., 316
Kim, K., 21 Kondratick, C. M., 155, 195, 206, 207,
Kim, N., 139, 148 217, 274
Kim, S. J., 21 Kong, X. P., 252
Kim, S.-R., 206, 230, 232–235, 237, 239–241 Kooiman, P., 77
Kim, S.-T., 51, 77–79, 81, 83–85, 87–89, Kool, E. T., 215
111, 112 Koonin, E. V., 51, 112, 212, 230, 232,
Kim, Y. S., 93 267–269, 289
Kimura, M., 186 Kornberg, A., 150, 158
Kingston, R. E., 61 Kornberg, R. D., 59
Kinnucan, E., 291 Korolev, S. V., 293
Kinoshita, K., 182, 311, 313, 315, 317, 323 Kouchakdjian, M., 13
Kinoshita, T., 27 Kow, Y. W., 7, 9
Kinoshita, Y., 27 Kowalczkowski, S. C., 194
Kinsohita, K., 317 Kowalski, J. C., 51
Kinzler, K. W., 107 Koyoshi, S., 271
Kirchgessner, C., 308 Kozak, M., 187
Kirchhoff, T., 147, 149 Kraemer, K. H., 44, 184, 221, 320
Kirk-Bell, S., 207, 216 Krahn, J. M., 147
Kisker, C., 152, 155, 189, 220, 275 Krainer, A. R., 325
Kisselev, A. F., 122 Kramarczuk, I. H., 210, 211, 273
Kitadokoro, K., 77, 79 Kramata, P., 177
AUTHOR INDEX 351
Krangel, M., 308 Lam, K., 308

Krause, K., 317, 323 Lamarre, D., 23
Krauss, G., 53 Lamb, J., 185, 287
Krejci, L., 194 Lan, V. T. T., 168, 171
Krogan, N. J., 287 Landreau, C., 143
Krokan, H. E., 6, 10, 19, 25, 316, 317 Lane, D. P., 21
Kroth, H., 210, 211, 239, 241, 273 Lane, W. S., 7, 9
Kruas, B. R., 187 Langenbacher, T., 79
Krzeminsky, J., 177, 210, 273 Lansford, R., 308, 311, 312
Kubota, Y., 24, 297 Laokes, D., 174, 180, 181, 191, 194
Kucherlapati, R., 322 Larimer, F. W., 168, 186, 206, 230
Kucho, K., 75, 77, 91 Larsen, E., 25
Kufe, D., 23, 119 Lassus, P., 108
Kugelberg, E., 241 Laszo, A., 23
Kulaeva, O. I., 212, 230, 232 Latham, K. A., 7, 9
Kumagai, K., 267 Lau, A. Y., 6, 9–11
Kumaki, S., 316, 317 Lau, P. J., 155
Kume, H., 266 Lauder, S., 287
Kung, H. C., 55 Laval, J., 7
Kunitomi, N., 174, 180, 182, 191 Lavrik, O. I., 21
Kunkel, T. A., 19, 93, 139, 141, 147–149, Lawrence, C. W., 152, 167–176, 179,
151–153, 157, 168, 173, 184, 190, 206, 180, 185–191, 205, 206, 229,
208–212, 214, 221, 229, 233, 237, 238, 232, 281
269, 271, 320 Lawrence, N. A., 143, 158
Kunz, B. A., 170, 192, 193 Layton, J. C., 240
Kuo, C. F., 7 Lazebnik, Y. A., 26, 108
Kuppers, R., 323 Le, X. C., 9, 25
Kuramitsu, S., 77, 79 Lebecque, S. G., 312
Kuraoka, I., 122, 211 Le Cam, E., 194
Kurihara, N., 121 Lechler, T., 168
Kurimasa, A., 111 Le Deist, F., 308
Kuriyan, J., 243, 252 Lee, C., 311, 319
Kurosky, A., 7 Lee, J. H., 122, 211
Kusumoto, R., 60, 206, 207, 210, 211, Lee, J. W., 149
217, 269 Lee, M. S., 16
Kuwana, T., 115, 116 Lee, P. L., 206, 241
Kuzminov, A., 281 Lee, P. W., 114
Kwon, K., 14 Lee, R. M., 325
Kycia, J. H., 14 Lee, S. H., 16, 19
Lee, S. K., 18
Lee, W. I., 316, 317
L Leem, S. H., 149
Lees-Miller, S. P., 189
Labelle, M., 26 Legerski, R. J., 43, 53, 57
Lacroix-Triki, M., 27 Lehmann, A. R., 57, 184, 207, 216, 217,
Lagrazon, K., 286 219–221, 243, 320
Lahav, G., 125, 127 Lei, K., 119
Lai, W. C., 267, 274 Lemeur, M., 143, 308
Lain de Lera, T., 149 Lemontt, J. F., 168–170, 188
Lalani, E., 143, 183, 185 Lengauer, C., 107
352 AUTHOR INDEX
Lenne-Samuel, N., 241–243 Linke, S. P., 108

Leonard, G. A., 13 Linn, S., 43, 45, 53, 57, 64, 121, 151, 155,
Le Page, F., 24, 25, 194 156, 217, 219
Lerenthal, Y., 123 Lippard, S. J., 61
Lesca, C., 27 Lipps, G., 53
Lescasse, R., 289 Lipton, L., 26
Leugers, S. L., 206 Little, J. W., 280
Levin, D. S., 21 Liu, D., 91, 110
Levine, A. J., 125, 127 Liu, J., 87–90
Levine, A. S., 53, 206, 207, 212, 230, 232 Liu, M., 59
Levine, M., 47 Liu, Q., 113
Levrero, M., 117, 118 Liu, Y., 92, 113
Levy, D. L., 151, 155 Livingston, A. L., 16, 26
Levy, Y., 313, 317 Livingston, D. M., 23, 112
Li, B.-H., 56 Livneh, Z., 152, 168, 194, 206, 229, 239, 245,
Li, G., 189 249–252, 256, 281
Li, G. M., 120 Ljungquist, S., 18, 24
Li, J., 22, 114 Lloyd, R. S., 7, 9, 16, 19, 210
Li, L., 43, 53, 57 Loakes, D., 174, 180, 182, 191
Li, X., 6, 13, 22, 113, 114 Loeb, L. A., 2, 18, 27, 158
Li, Y., 155, 156, 194, 310 Loechler, E. L., 148, 242
Li, Y. F., 83 Loewith, R., 189
Li, Z., 167, 168, 171–173, 183, 187, 190, 297, Logie, C., 61
310, 316, 321, 322 Lohman, P. H., 168, 171, 207, 216
Liakopoulos, D., 292 Lombardo, M. J., 206, 241
Liao, W., 325 Longley, M. J., 148, 152, 156, 157
Liao, X., 291 Lopes, M., 298
Liberi, G., 298 Lopez de Saro, F. J., 242, 243
Lick, K., 151 Lopez-Fernandez, L. A., 147
Lieber, M. R., 22, 149, 267, 308, 310–312 Lopez-Garcia, J., 28
Lieberman, J., 26 Lorch, Y., 59
Lim, D. S., 111, 112 Lord, J. M., 122
Lim, S. E., 152 Losson, R., 28
Lima, C. D., 292 Loukili, N., 289
Lin, C., 90, 91, 93, 94 Louneva, N., 177, 210, 273
Lin, D., 322 Lovering, R., 285
Lin, D. P., 121 Lowe, S. W., 108, 115
Lin, J., 112, 122 Lowery, D. M., 112
Lin, J.-J., 51 Lowndes, N. F., 112
Lin, P., 189 Lozano, J. J., 289
Lin, S. L., 289, 297 Lu, A. L., 6, 13, 16, 19
Lin, W., 168, 173, 186 Lu, C., 249
Lin, Y. C., 113 Lu, R., 7
Lindahl, T., 1, 2, 6, 16, 18, 21–25, 145, 147, Lu, X., 53
175, 182, 211, 297, 312, 316, 322 Lubratovich, M., 114
Lindsay, H. D., 110 Lucchini, G., 155
Lindsey-Boltz, L. A., 43, 45, 57, 64, 110 Lucey, M. J., 28
Lindsley, J. E., 248 Ludlow, C., 168
Ling, H., 152, 153, 212–215, 233, 234, Lukas, C., 115
236–238, 244, 245 Lukas, J., 106, 107, 113–115, 122
AUTHOR INDEX 353
Luk-Paszyc, M. J., 155, 189, 220, 275 Marapaka, P., 7

Luna, L., 9 Marcu, K. B., 317, 323
Lundgren, K., 114 Marenstein, D. R., 19, 25
Luo, C., 114 Margison, G. P., 25
Luo, G., 113, 122 Margosiak, S., 114
Margossian, L., 27
Margot, A., 194
M Marin, M. C., 117
Marini, F., 139, 143, 148, 155, 183, 185
Ma, L., 289 Market, E., 312, 316, 318
Ma, Y., 308 Markovina, S., 23
MacCallum, R., 152, 155 Marodi, L., 316, 317
MacDonald, J., 176, 179, 191 Marot, D., 118
Macdougall, E., 25 Marr, M. T., 57
MacFarlane, A. W., IV, 79 Marriott, D., 287
MacGinnitie, A. J., 324 Marsin, S., 24
MacGlashan, D., 47 Marth, J. D., 143
Maciejewski, M. W., 19 Martin, A., 313, 315, 316, 322, 323
Madhani, H. D., 287 Martin, S., 285
Madsen, P., 324 Martinez, A. C., 149
Madura, K., 287 Martini, E. M., 299
Maekawa, T., 60 Mas, P., 93
Maenhaut, M. G., 230, 233, 237, 241 Maser, R. S., 112
Maenhaut-Michel, G., 230, 240 Masson, M., 24
Maga, G., 137 Masuda, K., 189
Magnac, C., 317 Masuda, Y., 155, 168, 173, 189, 220, 275
Mahajan, K. N., 149, 310 Masui, R., 77, 79
Mahajan, R., 291, 292 Masumura, K., 25
Maher, E. R., 26 Masutani, C., 53, 60, 61, 141, 143, 153, 180,
Maher, V. M., 167, 168, 171–173, 187, 189, 206–212, 215–217, 219, 220, 221,
190, 220, 221, 297 233, 239, 269, 273–275, 321
Mahoney, W., 16, 19 Masuyama, S., 297
Majka, J., 110 Mataga, N., 79, 84, 85
Mak, T., 26 Matas, D., 24
Mak, T. W., 114 Mathews, C. K., 208
Makhov, A. M., 111 Mathis, D., 143, 308
Maki, H., 18, 248 Matsuda, T., 153, 184, 208–212, 221, 269,
Malhotra, K., 77, 81, 85, 87, 88 271, 320
Malik, S., 308 Matsui, K., 206, 230, 232–235, 237, 239–241
Malim, M. H., 325, 326 Matsumoto, Y., 21
Malone, M. E., 6 Matsunaga, T., 53, 121, 168, 171
Malynn, B., 310 Matsunga, T., 77, 87
Mangeat, B., 326 Matsuoka, S., 112, 113
Manis, J. P., 308, 311, 312, 318, 322 Matunis, M. J., 292
Maniwa, Y., 110 Matuschewski, K., 292
Manke, I. A., 112 Maymon, M., 93
Mannino, J. L., 121 Maynard, J., 26
Mao, C., 256 Mayne, L. V., 44, 57
Maor-Shoshani, A., 206, 239, 249, 250, Mazumder, A., 7, 18
252, 256, 281 McAuley-Hecht, K. E., 13
354 AUTHOR INDEX
McBride, K. M., 317 Michelson, R. J., 102, 109

McCarthy, J., 317, 323 Mihara, M., 115, 116
McCormick, J. J., 171, 220, 221, 297 Mikami, Y., 9, 10
McCulloch, S. D., 141, 153, 210, 211 Miki, K., 77, 79
McCullough, A. K., 7, 16 Miki, Y., 23
McDaniel, L. D., 57 Millar, C. B., 25
McDonald, J. P., 143, 147, 148, 184, 194, Miller, H., 177, 210, 211
206–210, 212, 216, 221, 230, 232, 321 Miller, J. H., 14
McDonald, W. H., 152 Miller, J. K., 13
McElhinny, S. A., 153 Milligan, D., 16
McEntee, K., 242, 249 Mills, M., 122
McFadden, G., 75 Milstein, C., 308, 316, 320, 322
McGill, C. B., 184 Milyavsky, M., 23, 24
McGrath, J. P., 286 Mimura, J., 266
McGregor, W. G., 167, 168, 171–173, Minko, I. G., 210
187, 190 Minowa, O., 25
McHenry, C. S., 141, 150 Mirzoeva, O. K., 122
McKee, R. H., 169, 170, 185, 188 Mishiro, S., 18
McKenna, S., 284, 285, 289–291, 297 Missura, M., 53
McKenzie, G. J., 206, 241 Mitchell, B. S., 149, 310
McKeon, F., 117 Mitchell, D. L., 60, 189, 221
McKeown, C. K., 24 Mitomo, K., 23
McManus, T. P., 167, 168, 171–173, 187, 190 Mitra, S., 6, 7, 9, 18–20, 23, 192
McMurray, C. T., 122 Mittal, S., 293
McNair, S., 220 Mittelman, L., 123
McRee, D. E., 7 Miura, A., 27
Mehta, A., 325 Miura, Y., 267
Meijer, M., 60, 189 Miyamoto, Y., 94
Meira, L. B., 102 Miyazaki, J., 9, 25, 94, 96
Melchers, F., 312 Mizukoshi, T., 61, 88, 212
Melchior, F., 291, 292 Mizuta, R., 308
Melino, G., 117, 118 Mo, J., 61, 64, 91
Mellon, I., 44, 56 Moch, C., 289
Melo, J., 102, 110, 113, 114 Mochan, T. A., 112
Memisoglu, A., 23 Mochida, S., 112
Mendelman, L. V., 172, 208 Mockler, T., 93
Mendelsohn, M., 308 Modrich, P., 156
Menissier-de Murcia, J., 24, 316 Moerschell, R. P., 180
Mer, G., 112 Moggs, J. G., 53, 55, 60
Merlo, P., 118 Mol, C. D., 7, 10, 15, 19, 20, 110, 192
Merson-Davies, L. A., 6 Moldovan, G. L., 155, 187, 219, 243, 274,
Messmer, B. T., 323 288, 292–294, 297, 298, 300
Metheny, L. J., 325 Molina, J. T., 21
Metzger, S., 291, 292 Moll, U. M., 115, 116
Meyerhans, A., 325 Moller, S. G., 93
Miao, G., 18, 23 Mombaerts, P., 308
Michael, D., 125 Moncalian, G., 122
Michael, H., 26 Monden, Y., 25
Michaels, M. L., 7 Monick, M. M., 23
Michel-Beyerle, M. E., 79 Moolenaar, G. F., 51
AUTHOR INDEX 355
Moore, D. H., 26 Myers, T. W., 173, 190

Moorthy, N. C., 23 Myrnes, B., 6
Moraes, T. F., 284, 285, 289–291 Myung, K., 155
Morales-Ruiz, T., 27
Moras, D., 232
Moreau, P. L., 248 N
Morelli, C., 168
Morgan, I. M., 220 Nadal, M., 289
Morgan, S. E., 118 Naderi, S., 108, 116
Morgan, W. F., 122 Nadji, S., 81, 82
Mori, K. J., 323 Naegeli, H., 53, 63
Mori, S., 113 Nagaoka, H., 317, 323
Mori, T., 53, 121 Nagawawa, H., 186
Morikawa, K., 9, 10, 234, 235, 239 Nagendran, V., 316, 317
Morin, L., 289 Nairn, R. S., 43
Morioka, H., 53, 121 Najarian, M. T., 27, 170, 193
Morita, R. Y., 240 Naka, K., 114
Moriya, M., 6, 177, 210, 211 Nakabeppu, Y., 25
Moriyama, K., 174, 180, 191 Nakada, S., 119
Morland, I., 9 Nakagawara, A., 267
Morrison, A., 168 Nakai, S., 25
Morrison, D. K., 119 Nakamura, H., 88
Morrison, J. R., 323 Nakanishi, M., 114
Morrow, J. S., 112, 114 Nakata, A., 248, 281
Moshous, D., 308 Nakatani, Y., 122
Mossmann, H., 143 Nakatsuru, Y., 266
Moustacchi, E., 168–170 Nakayama, K., 23
Moyal, L., 123 Nambu, Y., 319, 323
Mu, D., 43, 53, 55, 59 Nanjangud, G., 323
Mueller, M., 93 Naoi, Y., 168, 171, 186
Muijtens, M., 9, 25, 94 Napolitano, R., 239, 241, 242, 250
Mukhopadhyay, D., 324, 325 Nash, H. M., 7, 9
Mullen, G. P., 19 Nash, R. A., 24, 297
Mullenders, L. H., 44, 57, 216, 217 Natarajan, A. T., 44, 57
Muller, J. G., 6 Navaratnam, N., 323, 324
Muller, S., 24 Navarro, S., 267
Muller, W., 316 Navas, T. A., 155
Mungall, A. J., 168 Nealon, K., 21
Murakumo, Y., 155, 168, 180, 187, 189, Nebel, S., 118
220, 275 Nebert, D. W., 265
Muramatsu, M., 182, 311, 313, 315, 317, 323 Negishi, H., 116
Murante, R. S., 167, 171–173, 190 Negishi, K., 174, 180–182, 191, 194
Murli, S., 250 Negrini, M., 168
Murphy, M., 111 Nehme, A., 118
Murre, C., 323 Nelson, J. L., 206
Murthy, K. G., 23 Nelson, J. R., 167, 171–176, 179, 188–191,
Musacchio, A., 155 205, 232
Muto, T., 313, 317 Neuberger, M. S., 143, 157, 182, 183,
Muzi-Falconi, M., 298 185, 194, 312, 315, 316, 319, 320,
Myers, P., 114 322, 325, 326
356 AUTHOR INDEX
Neumeister, P., 323 O

Neuwald, A. F., 112
Newmeyer, D. D., 115, 116 Oakeley, E. J., 27
Ng, H. H., 15 O’Brien, P. J., 11, 17, 45
Ng, J. M. Y., 53 O’Brien, T., 169, 170
Ng, W. O., 77 Ocampo, M. T., 25
Nguyen, A., 112 Ochs, H. D., 316, 317
Nguyen, B., 114 Ochs, R. L., 112
Nguyen, D., 157, 168 O’Connell, M., 112
Nguyen, T. D., 60 O’Donnell, M., 139, 150, 153, 206, 221,
Niall, H. D., 283 235–237, 239, 241–243, 245, 249–252,
Nicholl, I. D., 21 256, 273, 281
Nichols, A. F., 53 Oettinger, M. A., 308
Nicholson, D. W., 26 Offer, H., 23, 24
Nick McElhinny, S. A., 149, 310 Ogata, E., 18
Nicolas, A., 194 Ogawa, O., 271
Niedergang, C., 24 Ogi, T., 206, 266, 267, 269, 271, 273
Nielsen, H., 25 O’Grady, P. I., 191
Nikaido, O., 53, 77, 87, 121 Ogura, Y., 189, 275
Nilsen, H., 16, 25, 182, 312, 322 O’Handley, S. F., 7
Nimnual, A., 120, 122 Ohashi, E., 180, 189, 206, 210, 211, 220, 239,
Ninio, J., 47 269, 271, 273, 275
Nishijima, M., 267 Ohkuma, Y., 60
Nishimura, S., 25 Ohmae, M., 189
Nishishita, T., 18 Ohmori, H., 152, 168, 177, 180, 189, 206,
Nissen, K. A., 60 210–212, 220, 229, 230, 233, 237, 239,
Nisson, P. E., 168, 169, 171, 187 241, 266, 267, 269, 271, 273–275
Niwa, H., 94, 96 Ohtsuka, E., 9, 10
Noda, T., 25 Okada, A., 308
Nohmi, T., 25, 152, 168, 206, 229, 230, Okada, H., 114
232–235, 239–242, 248 Okada, T., 168, 185, 271
Noll, D. M., 13 Okada, Y., 114
Nomura, T., 77, 87 Okamoto, T., 53
Nonoyama, S., 316, 317 Okamura, T., 79, 84, 85
Nookala, R. K., 247 Okazaki, I. M., 315, 323
Norbury, C., 152, 155 Okazaki, T., 18
Norman, D. P., 9, 11, 12 Okey, A. B., 265
Noskov, V. N., 174, 180, 191, 194 Okumoto, D. S., 44, 56
Notarangelo, L. D., 313, 317 Okuto, H., 174, 180, 191, 194
Novina, C. D., 26 Olieric, V., 243, 245, 246
Nowicka, A. M., 173–175, 179, Oliver, J., 24
188, 191 Ollis, D. L., 139
Nuber, U., 285 Olmsted, E. A., 143, 158
Numata, S.-I., 155, 168, 187, 189, 275 Olsson, C., 311
Nunoshiba, T., 192, 193 Olsson, M., 6
Nurse, P., 155 Oltz, E., 308, 310
Nussenzweig, A., 308, 312 Ong, P., 53
Nussenzweig, M. C., 308, 312, 317, Onrust, R., 252
318, 320 Ookuri, T., 186
Nyberg, K. A., 102, 109 Opitz-Araya, X., 108
AUTHOR INDEX 357
Opperman, T., 250 Parlanti, E., 21

Oppezzo, P., 317 Parraga, M., 147
Orban, P. C., 143 Parris, T., 121
O’Reilly, N. J., 285 Parsons, J. L., 26
Oren, M., 115–117, 125 Parsons, S. H., 26
Orkin, S., 310 Partch, C. L., 94
Orntoft, T. F., 26 Parwaresch, R., 317, 323
O’Rourke, E. J., 14, 17 Pascucci, B., 21
Orren, D. K., 47, 49, 51 Pasqualucci, L., 323
Oryhon, J., 293 Pastink, A., 168, 171
Osborn, A. J., 110 Pastushok, L., 284, 285, 289–291, 297
O’Shea, C., 64 Pata, J. D., 153, 212, 213, 233, 234
Osley, M. A., 287, 299 Patel, D. J., 13, 63, 323
Ossovskaya, V. S., 189 Paterson, M. C., 207, 216
Otevrel, T., 310 Patil, C., 308
Otoshi, E., 87, 89 Paull, T. T., 122, 308
Otsuka, C., 174, 180–182, 191, 194 Paulovich, A. G., 189
Ouellette, L. M., 220 Paunesku, T., 293
O-Wang, J., 266, 267 Pavletich, N. P., 285, 291
Owen, B. A., 122 Pavlov, Y. I., 151, 174, 180, 184, 191, 221, 320
Özer, Z., 81 Payelle-Brogard, B., 317
Özgür, S., 91, 93 Payne, G., 77, 79, 83–85
Ozkaynak, E., 283 Paz-Elizur, T., 245
Pearl, L. H., 10, 245, 247, 252
Pedersen, L. C., 147
P Pediconi, N., 118
Pelanda, R., 308, 312
Paciotti, V., 155 Pellicioli, A., 155, 298
Paciucci, R., 289 Pennell, R. I., 27
Pagés, V., 153, 155 Pepper, E. D., 206, 240
Pakrasi, H. B., 77 Perricaudet, M., 118
Pal, B. C., 6 Perrin, L., 326
Pan, P. Y., 18, 23 Perry, J. R., 168, 186, 206, 230
Panaitescu, L., 171 Persinger, J., 61
Panayotou, G., 10 Peters, A., 308, 311, 312, 318
Pancoska, P., 115, 116 Petersen-Mahrt, S. K., 157, 315, 319, 322, 325
Pandey, P., 118 Peterson, C. A., 53, 61
Pannicke, U., 308 Peterson, C. L., 291
Pao, A., 265 Petes, T. D., 291
Papadimitriou, K., 110 Petit, C., 52, 94
Papaioannou, V., 308 Petrenko, O., 115, 116
Papavasiliou, F. N., 312, 316, 318 Petrini, J. H., 106, 112, 122
Pappin, D. J., 112, 285 Petruska, J., 172, 208, 221, 312, 318–320
Parikh, S. S., 9, 10, 17, 19 Petti, A. A., 93
Park, C.-H., 53 Pfander, B., 155, 187, 219, 243, 274, 288,
Park, H. J., 81, 82 292–294, 297, 298, 300
Park, H. W., 77, 78, 81 Pfeifer, G. P., 265
Park, J.-S., 57 Pham, P., 152, 153, 156, 212–214, 221, 233,
Park, M. S., 21 235–237, 239, 242, 249–252, 255, 256,
Parker, A., 16, 19 281, 312, 316–320
358 AUTHOR INDEX
Philippe, N., 308 233, 234, 243, 271–274, 285, 287,

Philipsen, A., 317, 323 288, 291, 293, 294, 297, 298
Phillips, A. M., 51 Prasad, R., 19, 21, 147, 148, 208
Phillips, B., 195, 219, 274 Preston, B. D., 2, 18, 143, 158
Phillips, R. K., 26 Price, D. H., 59
Phoenix, F., 28 Prince, M. A., 7
Phung, Q. H., 316, 322 Pritsch, O., 317
Picard, D., 118 Prives, C., 23, 24, 117
Picher, A. J., 147, 149 Protic, M., 293
Pickart, C. M., 183–185, 286, 287, Ptak, C., 289–291
289, 290 Pu, H., 27
Pierce, A. J., 156 Puri, P. L., 118
Piersen, C. E., 7, 19 Putnam, C. D., 9, 17, 102, 109
Pietrokovski, S., 51 Pyrowolakis, G., 155, 187, 219, 243, 274, 288,
Pigatto, F., 26 292–294, 297, 298, 300
Pillaire, M. J., 27
Pinaud, E., 311, 312, 316, 318
Pisani, F. M., 233–235, 239 Q
Plante, D. T., 94
Plebani, A., 313, 316, 317 Qiao, Y., 230, 239
Plevani, P., 155, 298 Qin, X. F., 308, 312
Plosky, B. S., 143, 153, 212, 214, 221, 233, Qiu, C., 15
234, 243, 321 Qiu, J., 21, 22
Poch, O., 232 Quah, S.-K., 170
Podust, V., 156 Quong, M. W., 323
Poirier, G. G., 23, 26
Polanowska, J., 122
Pomerantz, R. J., 326 R
Pommier, Y., 18, 26
Ponamarev, M. V., 157 Raams, A., 217, 219
Ponten, I., 239, 241, 273 Rabinovich, N., 212, 230, 232
Poon, K., 274 Rada, C., 182, 312, 316, 320, 322
Popoff, I. J., 119 Radic, M., 312
Popoff, S. C., 18 Radicella, J. P., 14, 17, 19, 24
Porter, A. C., 108, 116 Radman, M., 206, 207
Posnick, L. M., 27, 170, 193 Rahman, D., 112, 285
Potapova, O., 119, 237 Rai, K. R., 323
Pourquier, P., 18 Rajagopalan, K. V., 77
Pouryazdanparast, P., 241 Rajagopalan, M., 242, 249
Pouyet, J., 23 Rajewsky, K., 143, 183, 185, 271, 308,
Povirk, L. F., 149 312, 316
Pradhan, D., 112, 114 Rajpal, D. K., 175, 176, 190, 206
Prakash, L., 18, 45, 152, 155, 168, 169, Ramiro, A. R., 317, 318, 320
172–178, 185, 190–195, 206–212, Ramotor, D., 192, 193
214, 215, 217, 219, 229, 233, 234, Ramsden, D. A., 149, 153, 310
243, 271–274, 285, 287, 288, 291, Randall, S. K., 212, 230, 232
293, 294, 297, 298 Randrianarison, V., 118
Prakash, S., 18, 45, 152, 155, 168, Rangarajan, S., 156
172–178, 185, 190–192, 194, 195, Rangel, R., 317, 323
206–212, 214, 215, 217, 219, 229, Rapic-Otrin, V., 53, 207, 208, 216
AUTHOR INDEX 359
Rasio, D., 155, 168, 187, 189 Roegner-Maniscalco, V., 248

Rasmussen, H. H., 324 Roest, H. P., 297
Rasmussen, L. J., 27, 170, 193 Rogers, S. G., 18
Raspaglio, G., 21 Rogers-Fani, P., 311
Rathbun, G., 308, 310 Rognes, T., 9
Rathi, A., 121 Rogozin, I. B., 143, 184, 212, 221,
Rattray, A. J., 184 312, 318, 320, 321
Raya, A., 267 Roldan-Arjona, T., 27
Raynaud-Messina, B., 27 Rolink, A., 312
Reagan, M. S., 57, 59 Rolli, V., 24
Reardon, J. T., 43, 48, 52, 53, 55, 56, 63, Rolseth, V., 9
64, 81 Romeijn, R. J., 168, 171,
Reback, P. B., 287 185–187
Rebollo, J. E., 248 Ropp, P. A., 139, 148, 149
Rechkoblit, O., 153, 173, 177, 178, 210, 273 Rosbash, M., 93, 94
Recht, J., 287, 299 Rosenberg, S. M., 206, 241
Reddy, M. S., 194 Rosenfeld, N., 125, 127
Register, J., 114 Rosette, C., 119
Reha’k, M., 23 Rosewell, I., 25, 143, 183, 185
Reichlin, A., 308, 312 Rossi, O., 21
Reina-San-Martin, B., 317 Roth, J. R., 241
Reinberg, D., 53, 59 Roth, T., 155, 168, 187, 189
Reinbolt, J., 243, 245, 246 Rothman, P., 311
Ren, Y., 81, 82 Rotter, V., 23, 24
Reppert, S. M., 93, 94 Rouse, J., 110
Reuven, N. B., 206, 249–252, 281 Roush, A. A., 206, 207
Revert, F., 267 Roy, G., 23
Revy, P., 313, 316, 317 Roy, R., 7, 23
Reynaud, C. A., 143, 147, 157, 221, 310, Rudolph, F. B., 325
320, 321 Ruetsch, N., 312
Reynolds, P., 297 Ruhland, A., 169
Riabowol, K., 189 Ruiz, J. F., 147, 149
Richardson, J. A., 267, 274 Rupert, C. S., 79
Richmond, E., 291 Rupp, D., 43, 49
Rickman, D. W., 94 Rupp, W. D., 280
Rieger, R. A., 7, 9, 14 Russell, L. D., 267, 274
Roberts, C. J., 168 Russell, P., 152
Roberts, J. D., 173, 190 Ryan, K., 114
Roberts, J. W., 57 Ryo, H., 77, 87
Roberts, R., 241
Roberts, R. J., 81, 88
Roberts, V. A., 75, 77, 91 S
Robertson, K. A., 26
Robins, P., 25, 211 Sabariegos, R., 147
Robson, C. N., 19 Sabatino, R. D., 173, 190
Robzyk, K., 287, 299 Sablitzky, F., 311
Roche, H. R., 170 Saijo, M., 122
Roches, H., 192, 193 Saito, S., 114
Rodriguez, M. S., 112, 147, 292 Saitoh, S., 152
Roe, S. M., 245, 247, 252 Sakai, D., 60
360 AUTHOR INDEX
Sakai, T., 114 Scharff, M. D., 312, 313, 315–318, 322, 323
Sakai, W., 168, 171, 186 Scharz, K., 310
Sakamoto, A., 168, 171 Schatz, D., 308, 316
Sakiyama, S., 266, 267 Scheel-Toellner, D., 122
Sakumi, K., 25 Scheffner, M., 285, 292
Sakuraba, Y., 297 Schenten, D., 143, 271
Sale, J. E., 168, 183, 185, 194, 315, 325 Scherer, S. J., 121
Salles, B., 27, 280 Scheuermann, R. H., 248
Salmon, M., 122 Schiestl, R. H., 194, 298
Samaranayake, M., 312, 318, 320 Schlesinger, D. H., 283
Sampson, D. A., 292 Schmeits, J. L., 155
Sampson, J. R., 26 Schneider, J., 287
Samson, L. D., 6, 9–11, 23, 25, 27, 170, Schnitzlein, W. M., 75
193, 316 Schonthal, A. H., 108, 116
Sanadai, S., 174, 180, 191, 194 Schrader, C. E., 322
Sanal, O., 308, 313, 316, 317 Schreiber, V., 24
Sancar, A., 43–45, 47–49, 51–53, 55–57, 59, Schrock, R. D. III, 7, 9
61, 63, 64, 73, 75, 77–79, 81–85, 87–94, Schuffert, A., 139, 148
96, 110, 111, 121 Schuler, M., 115, 116
Sancar, G. B., 52, 77, 79, 81–83, 87 Schultz, L., 118
Sanchez, A., 7 Schultz, M. C., 23
Sanchez, Y., 155 Schultz, R. A., 57, 267–269
Sanchez-Pulido, L., 289 Schwartz, D. C., 291
Sancho, E., 289 Schwartz, M. A., 119
Sandell, L. L., 108 Schwartz, M. F., 112, 114
Saniger, M. L., 147 Schwarz, K., 308
Sankaranand, V. S., 311, 313, 323–325 Schwarz, S., 27
Sansom, O. J., 25 Schwarz, S. E., 292
Sanz, F., 289 Scott, J., 323, 324
Sarasin, A., 25, 194, 217, 219, 220, 320 Scully, R., 112
Sarbassova, D., 112 Seawell, P. C., 7
Sarker, A. H., 7, 9, 25 Sedgwick, B., 6
Sarkissian, T., 114 Seeberg, E., 9, 17, 25, 193, 316
Sarrot-Reynauld, F., 316, 317 Seed, B., 310
Sartorelli, V., 118 Sehested, M., 112
Sasaki, S., 116 Seidl, K., 310, 311
Sata, K., 77, 87 Seimiya, M., 267
Saurin, A. J., 285 Seitz, E. M., 281
Saus, J., 267 Seki, M., 139, 148
Saveliev, S., 255, 281 Seki, S., 7, 9, 23, 25
Savva, R., 10 Sekiguchi, J., 310
Sawada, J., 122 Sekiguchi, M., 25, 150, 248
Sawchuk, D., 308 Selby, C. P., 44, 47, 51, 52, 56, 57, 59, 91,
Sawt, W., 310 94, 96
Saxon, A., 311 Selfridge, J., 25
Sayegh, C. E., 323 Sengupta, S., 117
Sayer, J. M., 153, 177, 210, 211, 239, 241, 273 Seo, K. Y., 242
Schaffer, A., 183 Serwe, M., 311
Schar, P., 24, 28, 297 Seufert, W., 292
Scharer, O. D., 7, 9, 10 Shafer, B. K., 184
AUTHOR INDEX 361
Shalitin, D., 91, 93, 94 Singh, K. K., 16, 19

Shall, S., 24 Singh, M., 143, 158
Shannon, K. E., 114 Singhal, R. K., 21, 186
Sharief, F. S., 139, 148 Sinha, N., 81, 82, 248
Shaw, J., 26 Skaliter, R., 245
Shaw, S. J., 94 Skjelbred, C. F., 25
Shcherbakova, P. V., 151 Skuse, G. R., 325
Sheehy, A. M., 325, 326 Slayton, W. B., 158
Shen, B., 21, 22, 110 Slechta, E. S., 241
Shen, X., 206, 221, 235, 239, 241, 242, 249, Slupphaug, G., 10, 19, 25, 316, 317
250, 252, 255, 256, 273, 281 Smerdon, M. J., 16, 59, 60, 112
Sheng, W., 291 Smerdon, S. J., 114, 139
Sherman, F., 180, 286 Smeyne, R. J., 26
Shi, Q., 51 Smith, B. T., 237, 250
Shibata, T., 77, 79 Smith, C. A., 7, 44, 56, 77, 87, 88
Shibue, T., 116 Smith, G., 308
Shibutani, S., 6, 13, 239, 273, 274 Smith, H. C., 325
Shikazono, N., 168, 171 Smithies, O., 94
Shilatifard, A., 287 Smulson, M. E., 26
Shiloh, Y., 110, 111, 123, 125 So, A. G., 173, 190
Shimizu, A., 319, 323 Sobol, R. W., 21
Shimizu, M., 233–235, 239 Sofuni, T., 230, 233, 237, 241
Shimizu, Y., 53, 266 Sohail, A., 15, 312, 318, 320
Shimizu-Nishikawa, K., 186 Sonada, E., 308, 312
Shimodaira, H., 121 Song, F., 17
Shinagawa, H., 248, 281 Songyang, Z., 112
Shinkai, Y., 182, 271, 273, 308, 311, 313 Sonneveld, E., 185
Shinohara, A., 119 Sonoda, E., 168, 185, 271
Shinoura, Y., 206, 248 Sossou, M., 24
Shiromoto, T., 9, 25 Soulier, J., 112
Shishikura, T., 267 Soustelle, C., 194
Shivji, M. K., 143, 183, 185 Sowden, M. P., 325
Shock, D. D., 139, 208 Spadari, S., 137
Shoham, G., 14 Spangrude, G. J., 158
Shulman, M. J., 323 Spence, J., 286
Shyu, A.-B., 268 Spielmann, H. P., 63
Sibghat-Ullah, 52 Spira, A. I., 18
Siciliano, M. J., 9 Spitz, D. R., 23
Sidorkina, O. M., 7 Spivak, G., 44, 56
Sieber, O. M., 26 Spoonde, A. Y., 26
Siede, W., 113, 141, 205–207, 265 Spooner, E., 7, 9
Siegmann, M., 27 Spyracopoulos, L., 289–291
Sigal, A., 125, 127 Srinivasan, V., 75
Silbergleit, A., 185 Srivastava, D. K., 21, 148
Silvian, L. F., 152, 212–214, 233 Staden, R., 320
Simhadri, S., 177 Staknis, D., 93
Simon, J. A., 168 Stall, A., 308
Simpson, L. J., 168, 183, 185 Stamato, T., 310
Singer, B., 25 Stamp, G., 25, 143, 183, 185
Singh, G., 61 Stanewsky, R., 93, 94
362 AUTHOR INDEX
Stanley, R. J., 79 Sunnerhagen, P., 110

Stapleton, M. A., 51 Suo, Z., 234, 236, 237
Stark, G. R., 108, 116 Suski, C., 139
Stary, A., 217, 219, 220, 320 Sutton, M. D., 250
Stasiak, A., 251, 252 Suzuki, H., 114
Stavnezer, J., 311, 322 Suzuki, N., 239, 273, 274
Stavropoulos, P., 318, 320 Sved, J., 14
Stefanini, M., 57 Svoboda, D. L., 43, 55
Steinacher, R., 28 Swann, P. F., 19
Steinborn, G., 206, 248 Sweasy, J. B., 248
Steitz, T. A., 139, 153, 212, 213, 233, 234 Syvaoja, J. E., 151
Stelter, P., 187, 220, 243, 274, 294, 295, Szankasi, P., 168
297, 298 Szyklo, T. E., 114
Stephen, S. Z., 322
Stern, D. F., 112–114, 155
Stewart, G. S., 112 T
Stewart, J., 243
Stewart, V., 308 Ta, V.-T., 317
Stivers, J. T., 2, 6, 14, 16, 17 Taccioli, G., 308, 310
Stoiber, D., 116 Tada, Y., 266
Stoica, B. A., 26 Tagawa, M., 266, 267
St Onge, R. P., 110 Tainer, J. A., 7, 9, 10, 15, 17, 19–22, 75,
Storb, U., 182, 308, 311, 312, 318 77, 91, 110, 192
Storck, S., 147 Takahashi, J. S., 94
Stracker, T. H., 122 Takahashi, M., 168, 173, 189, 275
Strahl, B. D., 61 Takahashi, Y., 266, 292
Strathern, J. N., 170, 184 Takai, H., 114
Strauss, B. S., 241 Takano, H., 25
Strominger, J., 308 Takano, R., 94
Stubbe, J., 7 Takao, M., 9, 25, 94
Stucki, M., 21, 112 Takaoka, A., 116
Studwell-Vaughan, P. S., 242 Takata, M., 168, 183, 185, 194, 271, 315
Stukenberg, P. T., 242 Takeda, S., 168, 183, 185, 194, 271, 315
Stumpf, J. D., 206 Takemori, H., 77, 87
Suarez, M., 206, 207 Takenaga, K., 267
Sugai, M., 311, 313, 319, 323 Takeshita, M., 6, 194
Sugasawa, K., 53, 60 Takio, K., 206, 207, 217
Sugaya, E., 186 Tamada, T., 77, 79
Sugino, A., 149 Tamai, K., 112, 113
Suh, H., 308, 312 Tan, C.-K., 173, 190
Sullivan, M. J., 108, 116 Tanaka, A., 168, 171
Sumii, M., 168, 173 Tanaka, H., 110
Sun, B., 7, 9 Tanaka, K., 122, 271, 273
Sun, G., 119 Tang, J., 64, 119, 122
Sun, Q., 51 Tang, M., 206, 221, 235, 239, 242, 249, 250,
Sun, X., 23 252, 256, 265, 281
Sun, Y., 310 Tang, R. H., 92
Sun, Z., 112, 114 Taniguchi, T., 116
Sung, P., 119, 185, 194, 287, 288 Tarone, R., 322
Sunkara, S., 9 Tashiro, J., 311, 317
AUTHOR INDEX 363
Tateishi, S., 168, 185, 271, 297 Toh-e, A., 292

Taylor, A. M., 112 Tokushige, H., 217, 219
Taylor, E. R., 220 Tomer, G., 249, 250, 256
Taylor, I. A., 112 Tomicic, M., 19, 23, 26
Taylor, J. M., 323 Tominaga, Y., 25
Taylor, J.-S., 7, 77, 81, 82, 87–90, 153, 173, Tomkinson, A. E., 21, 149
175, 176, 178, 190, 210, 221, 235, 239, Tomlinson, I. P., 26
242, 249, 250, 256, 273 Tommerup, N., 324
Taylor, R., 21 Tompkins, J. D., 206
Taylor, S., 323 Tonegawa, S., 308
Taylor, W. R., 108, 116 Tong, A., 287
Tchaiko, P., 322 Torchia, J., 28
Tchou, J., 7 Tordo, N., 232
Teebor, G. W., 19, 25 Torpey, L. E., 172
Tempczyk-Russell, A., 114 Torres-Ramos, C. A., 243, 293, 294
Teng, B. B., 323, 325 Toth, E. A., 152, 212–214, 233
Terada, T., 316 Tournier, C., 120, 122
te Riele, H., 316 Trincao, J., 152, 212, 214, 233
Terrados, G., 149 Tripathy, D. K., 75
Texido, G., 316 Tritt, R., 26
Tezcan, I., 308, 313, 317 Troelstra, C., 57
Thayer, M. M., 7, 9 Trono, D., 326
Theis, K., 152 Trowsdale, J., 285
Thelander, L., 152 Trucco, C., 24
Thelen, M. P., 110 Truong, T., 119
Therese, S. M., 94 Tsai, K. Y., 117
Thiry, S., 27 Tseng, H. M., 149
Thoma, F., 60 Tsui, C., 290
Thomas, D. C., 173, 190 Tsuji, M., 230, 239
Thomas, H. J., 26 Tsunasawa, S., 180
Thompson, C., 94, 96 Tsuzuki, T., 25
Thompson, C. B., 157 Tsvetkov, L. M., 113, 114
Thompson, C. L., 91, 94 Tucker, J. D., 24
Thompson, L. H., 23, 24, 55 Turelli, P., 326
Thomson, J. B., 13, 292 Turley, H., 26
Thomson, T. M., 289 Turner, J., 206, 249, 281
Thornberry, N. A., 26 Turner, T. K., 120, 122
Thresher, R. J., 51, 91, 94, 96
Tian, M., 311, 312, 316, 318
Tini, M., 28 U
Tissier, A., 147, 148, 184, 194, 206–210,
216, 221 Udell, C. M., 110
Tjian, R., 47 Ueda, R., 212
Tobias, J. W., 287 Uematsu, N., 212
Toczyski, D. P., 102, 108, 109, 110, 113, 114 Ueng, L. M., 18
Toda, T., 152, 155 Ugazio, A. G., 313, 317
Todo, T., 75, 77, 87–91, 94, 96, 168, 206, Ulbright, T. M., 26
212, 229 Ullah, A., 312, 318, 320
Toh, E. A., 292 Ulrich, H. D., 155, 187, 220, 243, 274, 289,
Toh, H., 75, 77, 87, 91 291, 294, 295, 297–299
364 AUTHOR INDEX
Um, S. J., 28 Venere, M., 112

Unk, I., 155, 175, 191, 192, 195, 219, 274 Venezia, N. D., 118
Ünsal-Kacmaz, K., 43, 45, 57, 64, 111 Venkatasubrahmanyam, S., 287
Ura, K., 61 Verbeek, S., 316
Urich, T., 312 Verdine, G. L., 4, 7, 9–15, 17, 25
Usuda, S., 18 Vereault, A., 16
Usui, T., 122 Verhoeven, E. E. A., 51
Utsugisawa, T., 119 Verkaik, N. S., 168, 185
Uziel, T., 123 Verkerk, A., 94
Verkoczy, L. K., 183, 321
Verly, W. G., 7
V Vermeulen, W., 57
Vermey, M., 297
Vagas, E., 91 Verselis, S. J., 114
Vaillancourt, J. P., 26 Vidal, A. E., 19, 24, 216, 217
Vaisman, A., 184, 194, 208–211, 214, 221, 233 Vidanes, G., 113
Valencia, A., 289 Vijay-Kumar, S., 286
Valentine, M. R., 235–237, 239 Vila, M. R., 289
Vande Berg, B. J., 21, 139 Villalobo, E., 289
VanDemark, A. P., 285, 290 Villemain, J., 194
van de Putte, P., 51 Viskochil, D., 325
van der Horst, G. T. J., 9, 25, 94, 316 Visse, R., 51
van der Spek, P. J., 53 Vitaterna, M. H., 94
Vanderwiele, D., 191 Vlatkovic, N., 155, 156
Van de Velde, J., 77 Vo, A., 311
Van Gelder, R. N., 93, 94 Vogelstein, B., 107
van Gent, D. C., 168, 185, 308 Vojta, P. J., 139, 148
van Gool, A., 57 Volker, M., 216, 217
van Gurp, C. G., 297 Volkmer, E., 110
van Hoffen, A., 44, 57 von Borstel, R. C., 170
van Houten, B., 43, 51, 63, 148 von Hippel, P. H., 46
van Kesteren, M., 51 Vorum, H., 324
van Klaveren, J., 297 Vousden, K. H., 117
Van Komen, S., 194 Vreeburg, J. T., 297
van Leenen, D., 94 Vuillier, F., 317, 320
van Roijen, J. H., 297 Vuong, B. Q., 119, 122
van Rossum-Fikkert, S., 51
Van Sloun, P. P. H., 168, 185–187
van Zeeland, A. A., 44, 57 W
Varghese, A., 308
Varlet, I., 185 Wada, Y., 168, 171, 186
Varley, J. M., 114 Wager-Smith, K., 93, 94
Varshavsky, A., 283, 286, 287 Wagner, J., 206, 219, 232–235, 237,
Vartanian, J. P., 325, 326 239–243, 245, 246, 249, 252
Vasconcelos, Y., 317 Wagner, R., 206
Vassylyev, D. G., 9, 10 Wahl, G. M., 108
Veaute, X., 194, 248 Wahrer, D. C., 114
Velasco-Miguel, S., 143, 267, 271, 274 Wain-Hobson, S., 325, 326
Venclovas, C., 110 Wakasugi, M., 53, 55, 121
Venema, J., 44, 57 Wakeham, A., 114
AUTHOR INDEX 365
Walker, D. R., 51 Weinert, T. A., 102, 109

Walker, G. C., 141, 168, 205, 206, 229, Weinfeld, M., 9, 25
230, 237, 248, 250, 265 Weiser, B., 206
Walker, L. J., 19 Weis-Garcia, F., 308
Wallace, J. D., 26 Weiss, B., 2, 7, 18
Wallace, S. S., 7, 9, 274 Weissman, A. M., 285
Walsh, C., 77, 82 Weitz, C. J., 93
Walworth, N., 113 Weller, S., 320
Wang, B., 112 Werling, U., 121
Wang, D., 61 Werner, R. M., 17
Wang, F., 18, 23 West, C. M., 108
Wang, G., 26, 291 West, M. G., 23
Wang, H. G., 23 Westcott, S. L., 112
Wang, J., 310, 317, 323 Weston, C. R., 119
Wang, J. Y., 108, 116–119, 121 Whitaker, L. L., 118
Wang, M., 175, 292 White, C. L., 143, 271
Wang, P., 285, 291 White, C. L. III, 267, 274
Wang, R., 119 Wibley, J. E., 10
Wang, R. P., 77, 90, 91, 94 Wie, Y.-F., 168
Wang, S. W., 152, 155 Wiederhold, L. R., 23
Wang, T. S., 110, 243, 275 Wierda, W. G., 317, 323
Wang, X., 43, 108, 116, 207, 216 Wiesendanger, M., 322
Wang, X.-D., 167, 168, 171–173, 187, 190 Wilker, E., 114
Wang, Y., 57, 121 Wilkinson, W. J., 286
Wang, Y. C., 221 Willer, D. O., 75
Wang, Y. Q., 267 Williams, B. L., 114
Wang, Z., 61, 147, 149, 151, 153, 168, Williams, B. R., 106, 122
173, 175–178, 186, 190, 206, 209–211, Williams, G. T., 26, 182, 312, 322
229, 273 Williams, K. R., 46
Warren, A. J., 43 Wilson, D. M. III, 7
Washburn, R. S., 57 Wilson, S. H., 16, 19, 21, 24, 139, 143,
Washington, M. T., 172, 173, 175, 176, 147, 148, 208
190, 206, 208–211, 215, 234, 271–273 Wilson, T. E., 149
Watanabe, M., 114 Winfried, E., 322
Watanabe, Y., 60 Wing, J., 220, 243
Waters, T. R., 10, 19 Winter, D. B., 184, 221, 316, 320, 322
Watkins, J. F., 288 Withka, J., 7
Watson, J. D., 158 Witkin, E. M., 44, 56, 248
Watson, W. P., 13 Wittenberg, C., 151
Weaver, D. R., 93, 94 Wittschieben, B. ., 64
Webb, B. L., 281 Wittschieben, J., 143, 183, 185
Wedekind, J. E., 325 Wolberger, C., 290
Weeda, G., 25, 316 Wolffe, A. P., 59
Wei, C. S.-J., 177 Wolgemuth, D. J., 207, 216
Wei, K., 322 Woloschak, G. E., 293
Wei, S. J., 23 Wong, E., 316
Wei, Y. F., 77, 90, 91, 94, 289, 297 Wong, I., 13
Weichselbaum, R., 118, 119 Wong, J. A., 114
Weill, J. C., 143, 147, 157, 221, 310, Woo, C., 322
320, 321 Woo, R. A., 114
366 AUTHOR INDEX
Wood, A., 287 Y

Wood, C. J., 323
Wood, L. D., 118 Yaffe, M. B., 112, 114
Wood, P., 316, 317 Yaffe, M. P., 286
Wood, R. D., 22, 23, 24, 43, 53, 55, 64, 139, Yagi, H., 153
143, 145, 147, 148, 183, 185, 211, 237 Yagi, T., 168, 185
Woodgate, R., 143, 147, 148, 152, 153, Yakovlev, A. G., 26
156, 168, 176, 179, 184, 191, 194, Yamada, A., 60, 206, 207, 215–217
205–217, 221, 229, 230, 232–239, Yamada, M., 206, 230, 232–235, 237,
241–245, 248–252, 255, 256, 273, 239–241
281, 321 Yamada, S., 182, 311, 313, 323
Woodring, P. J., 118 Yamaguchi-Iwai, Y., 185
Workman, J. L., 61 Yamaizumi, M., 168, 185, 217, 219, 271, 297
Worthington, E. N., 77, 91, 93 Yamamoto, K., 23, 87
Worthylake, D. K., 285, 288 Yamamoto, Y., 230, 233, 237, 241
Wright, C. L., 324 Yamanaka, S., 323
Wright, M., 27 Yamashita, Y. M., 271
Wright, P. M., 6, 13 Yan, S., 63
Wu, K.-J., 59 Yanagida, M., 112
Wu, M., 63 Yanai, H., 116
Wu, P., 15 Yang, A., 117
Wu, X., 22, 61, 147, 149, 153, 168, Yang, B., 326
173, 175, 176, 186, 190, 206, 209, Yang, D., 206
210, 273 Yang, D. D., 120, 122
Wu, Y. J., 91, 92 Yang, H. Q., 92, 117, 118
Wu-Baer, F., 286 Yang, I. Y., 210, 211
Wuerffel, R. A., 308, 312 Yang, I.-Y., 177
Wyatt, M. D., 6, 9–11, 25 Yang, K., 121
Wynshaw-Boris, A., 118 Yang, S. H., 18
Yang, W., 17, 152, 153, 212–215, 233, 234,
236–238, 244, 245
X Yang, X. P., 21
Yao, M. S., 122
Xanthoudakis, S., 18, 23, 26 Yaspo, M.-L., 143, 183, 185
Xiao, W., 27, 168, 280, 281, 284, 285, Yasuda, H., 292
288–293, 297–299 Yasuda, S., 267
Xie, Z., 149 Yasui, A., 9, 25, 77, 79, 94
Xin, H., 168, 173, 186, 206 Yates, J. R., 152
Xing, D., 7, 9 Yeiser, B., 240
Xing, J. Z., 9, 25 Yel, L., 316, 317
Xu, B., 111, 112 Yelent, B., 21, 22
Xu, J., 120, 122 Yin, J., 242
Xu, L., 311 Yoder, B. L., 293, 294
Xu, N., 268 Yodoi, J., 23
Xu, P., 25 Yokoi, M., 206, 207, 217
Xu, X., 113, 114 Yokota, Y., 319, 323
Xu, Y., 26, 118, 285 Yokoyama, S., 77, 79
Xu, Z., 26 Yonei, S., 9, 25
Xuong, N. G., 139 Yoshida, A., 26
Xu-Welliver, M., 24 Yoshida, K., 23
AUTHOR INDEX 367
Yoshihara, K., 26 Zhang, C., 326

Yoshikawa, K., 311, 315, 323 Zhang, D., 26
Yoshioka-Yamashita, A., 121 Zhang, H., 171, 326
Yoshizawa, I., 274 Zhang, J., 311
You, H. J., 2 Zhang, K., 81, 82, 311
Young, D., 189 Zhang, Q. M., 9, 25
Young, F., 308, 311 Zhang, X., 15, 208
Young, M., 243 Zhang, Y., 149, 153, 168, 173, 175, 177,
Yu, K., 311, 312 178, 186, 206, 209, 210, 273, 287
Yu, L., 267 Zhao, G. Y., 168, 185
Yu, S. L., 18, 215 Zhao, S., 77, 87–91, 93, 94
Yu, S.-L., 177, 190 Zhao, X., 77, 79, 87–91, 94
Yu, X., 93, 112 Zhao, Y., 243
Yuan, F., 149, 168, 173, 175, 186, 206, 209, Zharkov, D. O., 7, 9, 14
210, 273 Zheng, H., 43
Yuan, W., 286 Zheng, N., 285, 291
Yuan, Z. M., 118, 119 Zheng, Y., 27
Yuasa, M., 206, 207, 217 Zhou, B., 212, 213
Yudkovsky, N., 61 Zhou, B. B., 104, 110, 113, 114
Yurieva, O., 243 Zhou, B. L., 153, 233, 234
Zhou, J., 24, 155, 189, 220, 275
Zhou, T., 149
Z Zhou, Z., 155
Zhu, B., 27
Zabkiewicz, J., 25 Zhu, C., 23, 310
Zacharias, W., 171 Zhu, J., 118
Zaika, A., 115, 116 Zhu, J.-K., 27
Zajc, B., 177 Zhu, Y., 289
Zakian, V. A., 108 Zicha, D., 216, 217
Zan, H., 183, 321 Zimmer, D., 310
Zawel, L., 53 Zimmerman, E., 26
Zeng, X., 184, 208, 216, 221, 316, 320 Zinkel, R. A., 122
Zeugner, A., 93 Ziqiang, L., 322
Zgaga, Z., 194 Zou, L., 110, 111
Zhan, Q., 121 Zou, Y., 51
Zhang, B., 61 Zurer, I., 23, 24
SUBJECT INDEX
A domain structures of, 324, 325

E. coli, 317
Abasic site, 4, 7, 8, 18 expression regulation of, 323
adenine removal in, 193 gene conversion and, 315
bypass of, 175, 181, 191–192, 193, 206, immune specific processes and, 313
239, 249 mRNA and, 315, 323–324
dAMP insertion opposite, 192 mutant, double expression in, 316
dCMP insertion opposite, 192–193 mutation generation and, 319
deoxyuracil removal in, 193 mutations, two phase, 314, 321
enzymes replicating, 175 overexpression of, 323
lesions and residues of, 174–175 RNA substrate for in vivo, 315–316
mutagenesis induced by, 237 sequence specificity mechanisms of, 327
mutations induced by residues of, 192 SHM and, 313–316, 323
Pol IV bypass of, 239 SHM and activity separation of, 316–317
Pol bypass of, 175, 181, 193 SHM and DNA action of, 318
residues of, 174–175, 192 SHM initiation by, 317–318
Rev1p bypass of, 175, 181, 191–192 SHM mutations and, 183, 314, 320
UDG uracil action and, 18 SHM spectra stimulated by, 327
Action spectrum, 85 SHM transcription and, 312, 314
Activation-induced cytosine deaminase spliced forms of, 317
(AID), 157 substrate, 327
activity separation of, 316–317 wild-type, 317
antibody diversification and, 315 AhR. See Arylhydrocarbon receptor
B-cell transfection of, 315 AID. See Activation-induced
biochemical aspects of, 318–319 cytosine deaminase
cancer and overexpression of, 323 Antibody
cancer avoidance and SHM regulation AID and diversification of, 315
of, 323 CSR, 326–327
CLL expression of, 317 diversity of, 308, 315
cryptic activity of, 318 functional, 308
CSR and activity separation of, 316–317 mutagenesis and diversity of, 308
CSR initiation by, 317–318 SHM conversion of low-affinity to high-
CSR targeted by strand-breaks initiated by, affinity, 326
311–312 AP. See Apurinic/apyrimidinic
deamination dependent on, 318, APE. See Apurinic/apyrimidinic
319–320 endonuclease
deficiency, 313 Apobec proteins, 323–326
discovery of, 313 antiretroviral activity of, 325–326
DNA action of, 312, 318–319 Apobec 1, 324–325
DNA breakpoints and, 312 discovery of, 326
369
370 SUBJECT INDEX
Apobec proteins (cont.) plant development and enzymes of,

DNA deaminases, 324 27–28
E. coli overexpression of, 325 Pol and synthesis of, 147
mRNA tumor suppresser editing and, 325 Pol for, 142–148
retrovirus action of, 325–326 reaction, specialized and Pol function, 148
RNA deaminases, 324 SHM and, 314, 321–322
Apurinic/apyrimidinic (AP), 2 subpathways, 143–145
BER initiation and, 145 synthesis, 147
NEIL proteins containing proteins like, 25 uracil removal from DNA by, 316
Apurinic/apyrimidinic endonuclease (APE), XRCC1 binding to, 24
2, 18–19, 20 yeast, 147
cancer expression of, 26 BER. See Base excision DNA repair
DNA glycosylases and action of, 19 BRCT. See Breast cancer C-terminal domain
inactivation of, 26 Breast cancer C-terminal (BRCT) domain,
redox activity of, 19, 20 111–113
structural biology of, 19, 20 Burkitt’s lymphoma, 221
Arylhydrocarbon receptor (AhR), 265–266
Ataxia telangiectasia-like disease
(ATLD), 122 C
ATLD. See Ataxia telangiectasia-like disease
Autonomous pathway, 106, 123, 124–127 Cancer, 24. See also Breast cancer
cell death and, 125 C-terminal domain
checkpoint, 106, 124–125 AID overexpression and, 323
p53 in, 125 APE expression in, 26
BER and, 27
Chk2 kinases predisposition to, 114
B CS and, 45
DNA damage adaption and, 109
B cell DNA damage resulting in, 63
GC formation in, 310 DNA repair and, 101–102
MMR, 314, 322 genotoxic stress cell response in therapy
V(D)J recombination and maturation for, 102
of, 310 mismatched repair proteins and mutation
Base excision DNA repair (BER), 1–28 rate of, 120
cancer and, 27 p53 and, 102, 115, 117
chromatin substrate, 16 Pol and, 158
complexity of, 23 Pol and, 210–211
damage types repaired by, 5–7 Pol overexpression and, 267
disease and role of, 24–27 SHM/AID expression and avoidance
DNA glycosylase initiated, 145 of, 323
E. coli gap filling during, 145 skin, 45, 158
enzyme role, 27–28 TLS and, 168
enzymes, downstream, 18–23 UV-induced skin, 158
initiation of, 2 Cell cycle
knockout mice and, 24–27 apoptosis and, 108
long-patch, 147 checkpoint prolongation, 107–108
mammalian, 23–27 checkpoints on, 104, 105, 106, 112, 140,
MMR pathways overlapping with, 322 155, 299–300
p53 and, 24 DNA damage initiating checkpoints on,
PARP in, 21 104, 105, 106, 112
SUBJECT INDEX 371
eukaryotic cells and checkpoints on, Pol V lesion bypass and, 252
299–300 TLS and, 242
mismatched repair proteins, mutated and, Class-switch recombination (CSR),
120–121 308, 312
p53 withdrawal from, 116 AID activity separation for, 316–317
Pol and checkpoints on, 140, 155 AID initiation of, 317–318
transcriptional repression/genes of, 108 AID required for, 313–315
Cell death AID-initiated strand-break targeted,
adaptation-induced necrotic, 105, 109 311–312
AP and, 106, 125 antibody, 326–327
apoptotic, 108, 115–120 defective, 313
catastrophic, 105, 109 domain, constant, 310–311
clonogenic survival v. mitotic, 107–108 mechanism of, 311
DNA damage response and effectors of MMR deficiencies and, 322
apoptosis, 115–120 occurrence of, 309, 310–311
DNA damage-induced apoptotic, 116–117 Pol in, 320
fibroblasts and DNA damage induced region specific process, 311
apoptotic, 119 transcription, 309, 311–312
genotoxic stress induced, 105, 108 CLL. See Chronic lymphoid leukemia
mitotic, 106, 107–108 Cockayne Syndrome (CS), 44–45
necrotic, 105, 108, 109 Cognate clock proteins, 93
p53-dependent, 106, 108, 116, 125, 127 CPD. See Cis-syn cyclobutane dimers
passive, 107 Cryptochrome, 73–96
premature senescence, 108 biological world findings of, 75, 76
tyrosine kinases activated apoptotic, 118 V. cholerae, 96
UV-induced, 122 circadian clock regulation with, 94
Chromatin circadian photoreception and, 94
ATP-dependent remodeling complexes clock function of, 94–96
of, 61 cognate clock proteins bound to, 93
DNA transcription factors in, 61 cytoplasm combinatorial heterodimers in
protein effect of, 61 cytoplasm and clock function of, 96
repair, 59–62 definition of, 73
UV-induced damage distributed within, discovery of, 77
59–60 DNA binding, 91–92
Chronic lymphoid leukemia (CLL), 317 Drosophila, 93, 94
Circadian clock expression of, 91, 94
cryptochromes and regulation of, 94 family, 74
function of, 94 function, 92–96
mammalian, 94 growth and development in plants
UV light effect amelioration with, 91 regulated by, 91
Cis-syn cyclobutane dimers (CPD), 205 heterologously expressed, 91
Pol and lesion of, 215 human identified, 90, 91, 93
RAD30 bypass of, 207 isolation, 91
UV induced, 205 light-independent function, 94
clamp mammalian expression of, 94
E. coli polymerase interaction with, nucleic acid binding, 93
242–243, 244 as photoreceptor, blue light, 90, 94
mutagenesis and, 242, 248–249 photoreceptor function of, 92–93
Pol binding to, 242–247 photosensory function of, 93–94, 95
Pol IV modulation by, 234–235 phylogenetic trees of, 74–75, 76
372 SUBJECT INDEX
Cryptochrome (cont.) T:G mismatches in, 14–15

plant identified, 90 uracil residues in, 6
structure, 91–92 Y family Pol and replication of, 229
Synecocytis, 91 DNA bases
CS. See Cockayne Syndrome alkylation of, 6
CSA proteins, 57 canonical, 6
CSB proteins, 57, 59 hydrolytic deamination of amine-
CSR. See Class-switch recombination containing, 5–6
Cyclobutane pyrimidine dimer, 271 oxidation of, 6
ultraviolet light-induced damage to, 6–7
DNA binding
D ATP-dependent, 47
DDB protein, 64
Damaged DNA-binding protein (DDB), 53 kinetic proofreading, 47–48
DDB. See Damaged DNA-binding protein molecular mechanism of, 47
50 -deoxyribosephosphate (50 -dRP), protein, 121–122
2, 5, 208 DNA damage
DinB. See DNA damage inducible abasic residues as, 174–175
DNA adaption to, 105, 108–109, 125
AID action and, 318–319 adaptor proteins and signaling complexes
AID action and breakpoints of, 312 in, 111–113
backbone cleavage of, 2 adjacent damaged bases converted to
base pairs distinguished by abnormal dinucleotide adduct, 74, 87
protein of, 63 AP and repair activation of, 124
binding, cooperative of, 46–47 apoptosis induced by, 115–120
chromatin and transcription factors arrest response to, 64, 106, 107
of, 61 avoidance process of, 281
covalent linkage formed by, 266 biological responses to, 104–109
cryptochrome binding of, 91–92 cancer and adaption to, 109
fidelity, 279 cell-cycle checkpoints and sensor of,
histone interactions in, 61 188–189
initiator protein in, 63 cell-cycle checkpoints initiated on, 104,
lesion/protein binding and, 53 105, 106, 112
lesions’ interference with, 2 cellular effects, protection from of,
MutY bound to, 16 220–221
nucleosome-bound, 16 cellular response to, 102
nucleotide incorporation into, 239 detection specificity and, 52
PCNA and metabolism of, 293 DNA glycosylase recognition of, 10–16
Pol catalytic activities and, 219 DNA lesion level as cell response to, 102
Pol catalytic activities and, 219 DNA lesions and signaling in, 120–123
Pol crystal structure and, 214 DNA replication and, 152
Pol tolerance of geometric distortions effector kinases and responses to,
of, 214 113–114
Pol synthesis to short region, 209 eukaryote recovery and, 300
Pol IV and, 234 eukaryotic cell cycle arrest and, 64
protein binding of, 46–47 fibroblasts and apoptosis induced by, 119
protein phosphorylation and initiation genome, 307
of response to, 111 glycosylase recognition of, 10–15
SHM and AID action with, 318 irreversibility, 107–108
substrates for duplex, 15–16 mechanism of, 114–115
SUBJECT INDEX 373
mitosis blocked by, 106, 107 tolerance, 188, 279, 280

mm2 mutant and, 284, 289 transcribed strand repair, 56, 57
molecular components for initiating tyrosine kinases and response of, 118
response to, 109–115 UV-induced, 111
necrosis induced by adaptation to, in vitro repair, 61
105, 109 DNA damage inducible (dinB), 230
NER elimination of, 43, 44 biochemical properties of, 232–239
9-1-1 complex, 110 mutagenesis pathway dependent on, 237
objectives in field of response to, 127–128 phenotype, 230
p53 activated by, 106, 107 Pol activity of, 233
p53 and response of, 115–117 Pol IV as gene product of, 230–248
6-4 photolyase repair of, 88 Pol IV encoded by, 232–234, 239
photolyase repair of UV-induced, 73, 74 UmuC-like protein sequence homology
PIKK family of protein kinases and, shared with, 230–232
110–111 DNA glycosylases, 2, 4, 7–17
Pol bypass of UV-induced, 273 AAG structural family of, 9
Pol for replicating, 145, 152–155 activity, 18
Pol and, 273 adenine, 26
Pol integration in responses to, 189–190 amine nucleophile of, 4, 5, 7, 14
protein kinases activated by APE action and, 19
UV-induced, 111 BER initiated by, 145
proteins complexes induced by, 111 bifuctional, 2, 4, 5, 7, 16, 18, 23
proteins recognizing, 53 catalysis of, 16–17
PRR mutants and, 288 cytosine recognition in, 13
RecA and cellular response to, 248 damage recognition with, 10–16
RecA as sensor of, 281, 282 end-product inhibited, 16
recognition, 43, 45–48 HhH-GPD superfamily of, 9
repair pathways, 141–142 intrahelical recognition and removal
response coordination, temporal to, mode of, 11
104–109 lesions acted on by, 15
response coordination to, 101–128 mechanic classes of, 7–9
responses, 102–120, 123–127, monofunctional, 2, 7, 18
189–190, 248 MUG, 10
early, 107 MutM structural family of, 9, 11–13
immediate-early, 104–106 MutY recognition complex for, 13, 14,
late, 107 15, 16
results of, 63 oxidative lesions repair with
REV1p and tolerance of, 188 bifunctional, 23
S phase, 152 plant development and, 27
SAPK activated by, 119 recognition complexes for, 4, 10–15
search process in, 15–16 reducing agent in bifunctional,
sensor, 53, 110, 123, 188–189 4, 7–8, 12
signal transduction, 103, 109–110 structure of, 8, 9
signaling and repair proteins in, 120–123 substrate binding, 16
signaling mechanism, 104, 105, 120–123 substrate recognition, 14
SOS Pol response after, 280–281 T:G mismatches acted on by, 14–15
S-phase in response to, 106 thymine removal from, 10
temporal coordination of responses to, UDG catalysis of, 17
123–127 UDG structural family of, 9, 11–12
thermodynamic destabilization by, 63 uracil removal from, 10
374 SUBJECT INDEX
DNA lesions, 123 NHEJ double-strand break, 149

detection, 120 nucleotide transfer reaction and Y family,
DNA damage cell response and level 233–234
of, 102 PAD and Y family, 214
DNA damage signaling and, 120–123 PCNA function while bound to, 293
DNA replication through, 205 PCNA modifications and recruit of, 187
6-4 photolyase as UV light-induced, 86 Pol and Pol aligned to, 212, 213
Pol cognate, 273–274 Pol and Y family, 269
Pol substrates and, 176–177 Pol and REV1p in, 172
removal, 43 polymerization of, 139, 156
repair, 56 RAD30 protein and, 207
sensor, 123 repair, 2–3
signal transduction pathway and, replicative, 219, 232
109–110 Rev and Y family, 220
UV light-induced, 86 Saccharomyces cerevisiae, 137–139
DNA nucleobases, 1–2 SHM transcription and error-prone,
DNA polymerase (Pol), 19 312, 314
8-oxoG lesion bypass by, 210 sister chromatid cohesion, 151–152
Apobec proteins and deaminases of, 324 SOS, 242
BER, 142–148, 145–147 structure/composition, 139–141
Burkitt’s lymphoma and, 221 subdomains of catalytic subunits in,
cancer and, 158 139, 144
catalytic subunits, 139, 141 substrate preferences, 139–141
clamp binding by, 242–247 switch model, 249
crystal structure of Y family, 234 TLS, 144, 146, 152–153, 249
dinB and activity of, 233 TLS in vivo participation in, 241
diversity, 139 UV-induced DNA damage bypass by
DNA, 2–3 error-prone, 273
DNA damage replication with, 145, in vitro measure of, 270
152–155 Y family, 144, 153, 206, 208, 214, 220, 221,
DNA mismatched repair, 156 229, 232, 237–238, 269
DNA repair and, 137 yeast strain encoded, 151
DNA replication and, 137, 150–151 DNA polymerase V (Pol V), 248–257
DNA replication restart and, 155–156 biochemical properties, 250–255
dRP lyase activity, 138, 147 cellular, 230
dysfunction, 157–158 clamp and lesion bypass by, 252
E. coli, 137–139, 242–243, 244 E. coli, 229–257
error-prone, 145, 221, 229, 273, lesion bypass by, 250, 252–255
312, 314 RecA protein filament contact with,
families, 137–139, 140, 142 252, 254
functions of, 137–158 studies, 250–251
homologous recombination and, 155–156 TLS patch production by, 256
human, 137–139, 148–149, 157 UmuDC protein and, 248–257
human health and mutations in, 157 DNA polymerase IV (Pol IV)
immune system, 156–157 abasic site bypass by, 239
interstrand cross-link repair, 148–149 biochemical properties of, 232–239
lesion bypass and switch of replicative and cellular, 230
specialized, 255–257 clamp modulation of, 234–235
lesion bypass by, 153, 154, 205, 255–257 dinB gene, 230–248
NER, 143 DNA and, 234
SUBJECT INDEX 375
DNA primer template interaction with, insertion by, 210

245, 247 lesion bypass by, 273
DNA replication by, 239 little finger domain of, 213, 214, 215, 234
dNTP-stabilized misalignment and, localization of, 215–217
237, 238 mouse, 212, 215
E. coli, 229–257, 240–241 mutational specificity of, 314, 320
E. coli and induction of, 240 nuclei with foci containing, 216–217
extension, mismatched by, 236 nucleotide incorporation in, 210, 211–212
fidelity of, 235–237 palm domain of, 212–214, 215
incorporation, mismatched by, 236 PCNA interactions with, 219–220
induction of, 240 Pol and, 212
intracellular level regulation of, 240 Pol switch and, 219–220
lesion bypass by, 273–274 Pol, Y family aligned to, 212, 213
levels of, 240–241 regulation of, 215–217
little finger domain of, 245 Rev interactions with, 219–220
metabolism and, 240 template-binding complex formation
misinsertion capacities of, 235 and, 215
mutagenesis and, 235, 241–242 three-dimensional structure of, 212, 213
mutagenesis, lesion-induced and, 237 thumb domain of, 213, 214, 215
mutational spectrum and overexpression TLS by, 210–212, 217, 220
of, 237 truncated, 217–219
nucleotide incorporation catalyzed by, UV irradiation and cell, 216
233, 235 XP variants and mutations in, 217–219
pattern of, 233, 234 DNA polymerase (Pol ), 205–221
replication intermediate access regulation biochemical properties of, 207–210
to, 242–247 cell function of, 216
replisome, 240 DNA lesion miscoding potential and, 211
subdomains of, 243, 244, 245 DNA short region synthesis of, 209
TLS and, 240, 241–242 Drosophila, 212
in vivo expression level of, 239–240 duplication, 212
in vivo functions, 239–242 error-prone, 207
DNA polymerase (Pol ), 205–221 extension, mismatched of, 208–210
active site surface area of, 214 fidelity, 208
base insertion accuracy and, 211 foci localization of, 213, 217
biochemical properties of, 207–210 insertion by, 210
cancer and, 210–211 lesion bypass by, 273
catalytic core structure of, 212–215 localization of, 215–217
cell function of, 216 mispairs formed by, 208, 209
CPD lesion and, 215 mouse, 212
crystal structure of, 214 nuclear, 217
DNA and crystal structure of, 214 nuclei with foci containing, 216–217
DNA geometric distortion tolerance nucleotide incorporation in, 208, 210,
by, 214 211–212
DNA replication by, 215 PCNA interactions with, 219–220
Drosophila, 212 Pol and, 212
error-prone, 207 Pol switch and, 219–220
extension, mismatched of, 208–210 Pol, Y family aligned to, 212, 213
fidelity and crystal structure of, 214 regulation, 215–217
finger domains of, 213, 214, 215, 234 Rev interactions with, 219–220
foci localization of, 213, 217 SHM and, 221, 314, 321
376 SUBJECT INDEX
DNA polymerase (Pol ) (cont.) DNA damage response integration of,

TLS by, 210–212 189–190
UV irradiation and cell, 216 DNA lesions and substrates for, 176–177
DNA polymerase IV (Pol IV), 229–257 double-strand break homologous repair
DNA polymerase (Pol ) by, 185
C2HC zinc cluster sequence of, 271 double-strand break repair and, 184–186
cancer and overexpression of, 267 E. coli bypass by, 191
C-terminal, 269, 271 embryonic lethality and, 185
DNA damage and, 273 enzymatic studies with, 172–178
DNA lesions, cognate for, 273–274 enzyme structure and, 195
DNA repair and, 267 eukaryote function and, 167
enzymatic properties of, 271–274 extension, 177
enzyme activities of, 269–270 fidelity, 190–191
eukaroytic homologs of, 270, 271 function, 190
expression regulation of, 265–275 genetic analysis, 178–182
extension, 271–273 G-template specific, 174
gene structure of, 265–271 insertion and mutation frequency of,
human, 265–269 194–195
insertion, 271–273 insertion, lesion site of, 190
length, 268, 269, 270 insertion step, 177–178
lesion bypass by, 273, 274 lesion bypass frequencies, 177, 178–179
lesion substrates, 265–275 lesion-containing template in vitro studies
mouse, 265–269 of, 174–178
NLS in, 269 mutagenesis induced by, 170, 176
N-terminal, 269, 270 mutation, 179–180, 194–195
p53 inactivation and overexpression nucleotide incorporation fidelity of, 173
of, 267 nucleotide insertion, 178, 179, 191
PCNA preference by, 275 oligonucleotide template studies of,
Pol, Y family and, 269 174, 180
promoter region of mouse, 266 6-4 photolyase bypass by, 179
protein interactions, 275 properties of, 172–174
protein structure, 265–271 protein interaction and, 186–190
Rev1p and, 275 regulation of, 186–190
splicing variants in, 269–270 replication modes and, 187
testis expression of, 267 Revp and activity of, 172, 173–174
TLS SHM and, 182–184
in vitro by, 271 SHM caused by inaccurate, 182
in vivo by, 274–275 subunits of, 172
UV sensitivity of, 271–272 terminal mismatch extension efficiencies,
DNA polymerase V (Pol V) 172–173
E. coli, 229–257 TLS by, 167, 171, 185
TLS, 281 wild-type lesion bypass frequencies, 179
DNA polymerase (Pol ) XP and defects in, 206
abasic site bypass by, 175, 181, 184, 193 DNA repair, 46, 60–61, 298
B-family polymerase, 172 cancer and, 101–102
bypass frequency of mutant deficient, cell cycle checkpoints and activation
179–180 of, 103
cellular functions of, 167–195 cellular, 101
dCMP insertion, 173–174, 179–180, 182 importance of, 101–102
deoxycytidyl transferase activity, 174 mismatched, 156
SUBJECT INDEX 377
naked, 60–61 clamp interaction with Pol of,

pathways, 141–142 242–243, 244
Pol and, 137, 141–150, 156 DNA damage tolerance in, 280
Pol and, 267 dual incisions in, 49, 50
process, 279 excinuclease, 49–51, 52
PRR and, 279 excision nuclease, 49–51, 55–56
quantum yield in photolyase, 84 molecular matchmakers, 47
UV light effect amelioration with, 91 NER, 43–65, 49–52, 143
DNA repair polymerase (dRP), photolyase, 78, 86, 87
19–23 6-4 photolyase, 87
long-patch repair, 3, 19–23 Pol IV, 229–257, 240–241
PARP and reaction of, 21 Pol IV induction and, 240
Pol and, 138, 147, 148 Pol recognized in, 137–139, 153
short-patch repair, 3, 19–23 Pol V, 229–257
DNA replication, 150–151, 298 Pol bypass of, 191
base modifications and, 248 RecA protein in, 243, 299
DNA damage and, 152 repair pathway, 148
fork regression and, 155–156 replicative machinery in, 256
homologous recombination and, 156 transcription-coupled repair, 56–57, 58
immune system development and, 156 transcription-independent repair in, 50
lesion block of, 248 E2 proteins, 283–286
mitochondrial, 145, 152 Effector kinases, 113–114
PCNA clamp and, 110 cancer predisposition of Chk2, 114
Pol, 142, 150–151 Chk1, 113–114, 123–124
Pol , 215 Chk2, 114, 123–124
Pol IV, 239 DNA damage response and, 113–114
process, 279 FHA domain contained in Chk2, 114
stalling of, 256 knockout of Chk1, 113–114
strand, 140, 142, 151 knockout of Chk2, 114
UDG and, 25
DNA-dependent protein kinases
(DNA-PK), 118 F
DNA-PK. See DNA-dependent
protein kinases FHA. See Fork-head associated domain
dNTP. See Nucleoside triphosphate Fork-head associated (FHA) domain,
Drosophila 111–112
cryptochrome, 93, 94 Chk2 kinase containing domain of, 114
Pol , 212 interactions, 112–113
Pol , 212
dRP. See DNA repair polymerase
50 -dRP. See 50 -deoxyribosephosphate G
G ! A hypermutation, 325–326
E GASP. See Growth advantage in stationary
phase
E. coli GC. See Germinal centers
AID, 317 Genetics, 1, 102
Apobec overexpression in, 325 Genotoxic stress
BER and gap filling of, 145 apoptosis activation by, 108
Cho, 63 cell death induced by, 105, 108
378 SUBJECT INDEX
Genotoxic stress (cont.) L

cellular response to, 102, 103
p53 family member caused, 117–118 Lesion bypass
Germinal centers (GC), 310 oligonucleotide, 174, 180–181
Goodpasture antigen binding protein Pol, 153, 154, 205, 250,
(GPBP), 267 252–257, 273
GPBP. See Goodpasture antigen replicative and specialized switch
binding protein during, 255–257
Growth advantage in stationary phase Pol , 273
(GASP), 240 Pol , 273
Pol , 273
Pol specialized in, 255
H Pol V, 250, 252–255
Pol frequencies of, 177,
8-HDF. See 8-hydroxy7, 8-didemethyl-5- 178–179
deazariboflavin RecA, 251
High fidelity (Hi Fi), 312, 314 Rev1p frequencies of,
HIGM. See Hyper-IgM syndrome 178–179
HIV, 325–326 Lesions, 2
Human transcription termination factor 2 8-oxoG, 210
(TTF2), 59 abasic residues and,
8-hydroxy7, 8-didemethyl-5-deazariboflavin 174–175
(8–HDF), 77, 79 alkylated bases, 17
Hyper-IgM syndrome (HIGM), 313 DNA glycosylases acting on, 15
DNA glycosylases, bifunctional repair
of oxidative, 23
I DNA replication blocked by, 248
excision of, 59
ICL. See Interstrand cross links IS and increase of, 123
Ig. See Immunoglobulin gene mutagenesis induced by,
Immunoglobulin gene (Ig), 156 237–239
high-affinity, 182 NER of, 59, 63, 121
SHM induction in, 312 nontranscribed, 58
variant conversion of, 307 nucleobase, 2, 3
Integrative surveillance (IS), 106, oxidative, 22–23, 24
123–124, 126 oxoG, 6, 210
lesion increase and, 123 Pol IV and mutagenesis induced
regulatory hub of, 123 by, 237–239
Interstrand cross links (ICL), 148–149 Pol in vitro studies of templates
Ionizing radiation (IR), 116 containing, 174–178
ATM kinase activation with, Pol insertion and, 190
122–123 propanodeoxyguanosine, 177
mutagenesis induced by, 169 Rev1p in vitro studies of templates
p53 and, 127 containing, 174–178
IS. See Integrative surveillance Rev1p insertion and, 190
UV induced, 63, 91,
121–122
K in vitro assembly of nucleosomes
containing site-specific, 61
Kinetic proofreading, 47–48, 52 in vitro repair of oxidative, 24
SUBJECT INDEX 379
M Nucleobases, 1–2, 3
Nucleoside triphosphate (dNTP), 212
MagI. See 3-methyladenine glycosylase I Nucleotide excision repair (NER), 2, 43–65
Maltose binding protein (MBP), 249 damage recognition specificity with, 46
MEF. See Mouse embryo fibroblasts defects in, 43–44
Methenyltetrahydrofolate (MTHF), 73, 75, DNA damage recognition and, 43
77, 78 dual incisions in, 43, 48, 49, 50
5-methylcytosine, 14–15 E. coli, 43–65, 143
Mitosis-promoting factor (MPF), 104, 106 human, 43–65
MMR. See Postreplication human cell factors of, 63
mismatched repair kinetic proofreading, 47–48
Molecular matchmaker, 47 lesion removal with, 63
E. coli, 47 mechanisms of, 48–56
enzyme system use of, 52 Pol for, 143
Mouse embryo fibroblasts (MEF), 119–120 repair factors in human, 49, 52–53
MPF. See Mitosis-promoting factor resynthesis, 43
mRNA polymerase, 268 SHM and, 316
AID and, 315, 323–324 SNF and, 61, 62
Apobec-1 and tumor suppresser editing specificity of human, 56
by, 325 steps of, 43
Apobec-1 as editing enzyme of, 323–324 subpathways, 143
editing, 315, 325 substrate range, 55
tissue expression of, 269 SWI and, 61, 62
tumor suppresser editing by, 325 transcription coupled, 56
Mutagenesis transcription stimulated, 44
abasic site induction of, 237 transcription-independent, 50, 54
AID-induced, 323 UV sensitivity and, 43–44
antibody diversity and, 307 UV-induced lesions and, 121
clamp and, 242, 248–249 in vitro, 64
dinB-dependent pathway of, 237 in vivo, 64
genetic requirements of induced, 248–250
IR induction of, 169
lesion-induced, 237–239 O
Pol IV and, 235, 237, 241–242
Pol induction of, 170, 176 OxoG. See 8-oxoguanine
RecA protein and, 248–249 8-oxoguanine (oxoG), 6
SOS Pol and, 242 adenine paired with, 15
tumorigenesis and AID-induced, 323 MutM binding to, 12–13
UV light induction of, 168, 176, 237 MutY recognition mode, 13, 14
Pol bypass of lesion, 210
Watson-Crick face of, 12
N
NEIL proteins, 9, 25 P
NER. See Nucleotide excision repair
NHEJ. See Nonhomologous end-joining p53, 102
Nimejin Breakage Syndrome, 122 activation, 106, 107, 127, 267
NLS. See Nuclear localization signal AP and, 125
Nonhomologous end-joining (NHEJ), 149 apoptosis dependent on, 108, 116
Nuclear localization signal (NLS), 269 BER and, 24
380 SUBJECT INDEX
p53 (cont.) kinetic constants for reaction

cancer and, 115, 117 of, 79
cell death and, 127 monomeric proteins, 77
cells deficient in, 116 phylogenetic trees, 74–75, 76
DNA damage activation of, 106, 107 prokaryotic organisms possessing, 75
DNA damage response and, 115–117 quantum yield in, 84–85
domain, 115 quantum yield of DNA repair by, 84
genes regulated by family of, 117 reaction mechanism of, 79–86
identified, 115–116 structure, 77–79
IR and, 127 substrate binding, 79, 81–83
mutations, 265 substrate binding specificity of, 81, 83
negative feedback regulation of, 125–127 T. thermophilus, 74
PAH and mutations of, 265 three-dimensional diffusion substrate
phosphorylation, 115 binding, 81
Pol overexpression and inactivation vertebrates with, 75
of, 267 in vitro, 79
regulation, 125–127 in vivo, 79
related proteins, 117–118 6-4 Photolyase, 86–90
self-regulatory loop, 127 base-flipping mechanism, 88
transcription encoded factors and, 117 binding, 87–88
PAD. See Polymerase-associated domain catalysis, 88–90
PAH. See Polycyclic aromatic hydrocarbons DNA repair with, 88
PARP. See Poly ADP-ribose polymerase E. coli, 87
PCNA. See Proliferating cell nuclear antigen enzyme-substrate complex formation
PEO. See Progressive external and, 88
ophthalmoplegia flavin to substrate electron transfer and,
Photolyase, 73–96 89–90
A. nidulans, 74 photoantenna, 87
action spectrum and, 85–86 photolyases v., 90
aromaticity loss and, 82 photoproduct flipped out by enzyme
backbone distortion and, 81 bound DNA and, 88–89
binding free energy and positively charged Pol bypass of, 179
groove in, 83 Rev1p bypass of, 179
biological world findings of, 75, 76 three-dimensional diffusion and, 87
canonical pyrimidines split by dimer Photoreceptors, blue-light
radicals and, 84 autophosphorylating kinase activities
catalysis, 79, 80, 83–86 of, 93
crystal structure of decamer duplex and, cryptochrome as, 73–96, 90, 92–93
81, 82 photolyase as, 73–96
cycloreversion catalyzed by, 83 plant identified, 90
Dewar valence isomer photorepair with, 90 Phylogenetics, 74–77
dimer pyrimidine moieties in substrate Pol. See DNA polymerase
binding by, 82 Pol . See DNA polymerase
dimer splitting and, 84 Pol . See DNA polymerase
dinucleotide flipping and, 83 Pol IV. See DNA polymerase IV
discovery of, 77 Pol . See DNA polymerase
E. coli, 78, 86 Pol V. See DNA polymerase V
enzyme/substrate binding, 79, 83 Pol . See DNA polymerase
family, 74 Poly ADP-ribose polymerase (PARP),
induced-fit mechanism and, 81 21, 23–24, 26
SUBJECT INDEX 381
Polycyclic aromatic hydrocarbons (PAH), SUMO modification of, 292, 293,

265, 266 297–298
Polymerase-associated domain (PAD), 214 sumoylation of, 293–295, 298
Postreplication mismatched repair (MMR) TLS interactions with, 274
B cell, 314, 322 Ub and covalent modification of,
BER pathways overlapping with, 322 297–298
CSR and deficiencies of, 322 Protein kinases. See also DNA-dependent
SHM and, 314, 321–322 protein kinases
Postreplication repair (PRR), 279–300 ATM, 111
DNA damage and mutants of, 288 ATR, 111
error-free and error-prone, 298 JNK, 119–120
eukaryote, 281–283, 295–297 p38, 119–120
function, 283 PIKK family of, 110–111, 119, 123–124
future directions for studies of, 297 stress-activated, 119–120
Mms2-Ubc13-Rad5 and, 288–291 PRR. See Postreplication repair
pathways, 281–283 Pyrimidine dimer, 6–7
PCNA and, 292–295, 300
PCNA covalent modifications and,
292–295 Q
prokaryotes, 280–281
protein conjugation in, 286–292 Quantum yield, 84–85
Rad6-Rad-18 and, 286–288 DNA repair by photolyase, 84
RecA in, 280–281 Förster radiationless energy transfer
reconstitution, 297 mechanism efficiency and, 84–85
Ub conjugation in, 291 interchromophore distance efficiency
Ubc9-Sizl and, 291–292 and, 85
Progressive external ophthalmoplegia photoantenna/catalytic cofactor energy
(PEO), 157 transfer, 84
Proliferating cell nuclear antigen
(PCNA), 219
covalent modification of, 294, 295, R
297–298
desumoylation, 300 RAD5 homologs, 295–296, 298–299
DNA catalytic activities increased by, 219 RAD30 genes
DNA metabolism and, 293 CPD bypass by, 207
eukaryotic organism conservation by, identification of, 206–207
293, 297 Pol activity of, 207
eukaryotic replication machinery and, postreplication repair pathway
292–293 and, 207
Lys63 polyubiquitination of, 298, 299 template-dependent reaction and, 207
modification, 280 UV light and disruption of, 207
monoubiquitinated, 220 XP and, 207
Pol bound, 293 Reactive oxygen species (ROS), 6
Pol interactions with, 219 RecA protein
Pol preference of, 275 DNA damage sensor, 281, 282
PRR and, 292–295, 300 DNA damage tolerance in, 280, 281
PRR via covalent modifications of, E. coli, 299
292–295 filament, stabilized of, 252
Rad6 -Rad18 complex targets, 288 lesion bypass and, 251
SUMO conjugation to, 298 mutagenesis and, 248–249
382 SUBJECT INDEX
RecA protein (cont.) nucleotide insertion by, 179

Pol V contact with filament of, 252, 254 oligonucleotide template studies of,
targets, 300 174, 180
Recombination signal sequences 6-4 photolyase bypass by, 179
(RSS), 308 Pol and, 275
Repair proteins Pol motifs in, 172
cancer cell mutations and production levels, 187
mismatched, 120 properties, 172–174
cell cycle and mutated mismatched, protein interaction and regulation of,
120–121 186–190
DNA damage signaling and, 120–123 regulation, 186–190
mismatch, 120–121 replication and mutant, 188
MRE11–RAD50 complex, 122–123 SHM and, 182–184, 183
mutated mismatched, 120–121 subunits, 172
Rev proteins (Revp) terminal mismatch extension efficiencies,
abasic site bypass by, 175, 181, 172–173
191–192, 194 tetrahydrofuran bypass frequencies of,
B-family polymerase, 172 181–182
cellular functions of, 167–195 TLS, in vivo and, 275
chromosome aberration frequencies transcript level increase in, 186–187
in, 185 transferase activity, 194
competitive binding to, 189 wild-type lesion bypass frequencies
dCMP insertion, 173–174, 178, 179–180, of, 179
182, 194, 220 Revp. See Rev proteins
deoxycytidyl transferase activity of, RNA polymerase, 57
174, 188 AID, in vivo and substrate of,
DNA damage tolerance and, 188 315–316
domain structure of, 168, 169 Apobec proteins and deaminases of, 324
double-strand break repair and, 184–186 recognition by proxy with, 57
double-strand break-induced mutations SHM and, 183
of, 184 RSS. See Recombination signal sequences
double-strand breaks and disruption of,
185–186
enzymatic studies with, 172–178 S
enzyme structure and, 195
enzymes recruiting, 186 SAPK. See Stress activated
eukaryote function and, 167 protein kinases
functions of, 170–171, 173–174, 190 SCID. See Severe Combined Immune
functions of yeast, 173–174 Deficiency
genetic analysis of, 178–182 SCN. See Suprachiasmatic nuclei
G-template specific DNA polymerase, 174 Severe Combined Immune Deficiency
homology, 206 (SCID), 111
insertion and mutation frequency of, SHM. See Somatic hypermutation
194–195 Small ubiquitin-like modifier (SUMO),
insertion, lesion site of, 190 280, 291
lesion bypass frequencies, 178–179 PCNA and conjugation of, 298
lesion-containing template in vitro studies PCNA covalent modification and,
of, 174–178 297–298
mutation, 169–170, 184, 188, PCNA modification with, 292, 293
194–195 Ub charged surface and, 292
SUBJECT INDEX 383
SNF, 61, 62 strategy of, 292

Somatic hypermutation (SHM), 157, Ub and, 294–295
313–323 Suprachiasmatic nuclei (SCN), 94
AID activated mutation process, 314, 320 SWI, 61, 62
AID activity separation for, 316–317 Synecocytis, 91
AID and, 183, 312, 313–317, 320
AID initiation of, 317–318
AID required for, 313–315 T
AID stimulated spectra of, 327
AID/DNA action during, 318 T. thermophilus, 74
antibody conversion from low-affinity to TCR. See Transcription-coupled repair
high-affinity with, 326 TDG. See Thymine DNA glycosylases
antibody diversity and, 307–312 TdT. See Terminal deoxynucleotidyl
antigen binding and, 307–308, 309, 310 transferase
antigen recognition and, 307–308 Terminal deoxynucleotidyl transferase
B-cell line frequency of, 182 (TdT), 156–157, 308–310
biochemical perspective of, 326–327 3-methyladenine glycosylase I (MagI),
cancer avoidance and AID expression 13–14
regulation in, 323 30 -untranslated region (30 -UTR),
definition of, 182 268–269
Hi Fi and, 312, 314 30 -UTR. See 30 -untranslated region
Ig induction of, 312 Thymine DNA glycosylases (TDG), 28
mutation process, 314, 320 TLS. See Trans-lesion Synthesis
mutational panacea, 307–327 Transcription-coupled receptors, 56–59
NER pathway and, 316 E. coli repair, 58
Pol human cell repair, 60
error-prone and, 312, 314 Transcription-coupled repair (TCR), 64
inaccurate produced, 182 Transcription-repair coupling factors
Pol and mechanism of, 221 (TRCFs), 44, 56–57, 60
Pol and, 221 Translesion Synthesis (TLS), 152, 205
Pol and, 221 cancer and, 168
Pol and frequency of, 314, 321 clamp and, 242
Pol in, 182–183, 182–184, 221, 312, 314, (mis)incorporation, 205
320–321 patch, 256
Pol mRNA levels during, 183 PCNA interactions with, 274
Rev1p in, 182–184, 183 Pol, 144, 146, 152–153, 155, 167, 171, 178,
transcript levels during, 183 185, 241, 249, 255–257
transcription required by, 311–312, 313 replicative and specialized switch
transition mutations of, 318–319 during, 255–257
two-phase process of, 314, 319–321 Pol , 210–212, 217, 220
Stress activated protein kinases (SAPK), Pol , 210–212
119–120 Pol in vivo participation in, 241
DNA damage activated, 119 Pol IV and, 237, 240, 241–242
gene expression regulated by, 119 Pol in vitro, 271
JNK as, 119–120 Pol in vivo, 274–275
p38 as, 119–120 Pol V-mediated, 252, 281
SUMO. See Small ubiquitin-like modifier Pol-clamp interaction with, 144, 155
Sumoylation, 279–300 Rev1p, 167, 178, 275
PCNA, 293–295 Rev1p and in vivo, 275
PCNA conjugation and, 298 SOS Pol and, 242
384 SUBJECT INDEX
Translesion Synthesis (TLS) (cont.) NER correction of lesions induced by, 121
steps of, 205 6-4 photolyase as DNA lesion induced
successful, 255 by, 86
UmuC protein and, 249 photolyase repair of DNA damaged by,
TRCF. See Transcription-repair coupling 73, 74
factors Pol and irradiation of, 216
Trichothiodystrophy (TTD), 45 Pol and irradiation of, 216
TTD. See Trichothiodystrophy Pol sensitivity to, 271–272
Tyrosine kinases, Abl, 118–119 Pol and mutagenesis induced
apoptosis activated by, 118 by, 176
cell adhesion and activation of, 119 RAD30 disruption and, 207
DDB and, 122 XP and mutation spectra of, 221
nuclear, 118 UmuC genes, 281
UmuD genes, 281
Uracil-DNA glycosylase (UDG)
U DNA glycosylase, 9, 11–12, 17
DNA replication and, 25
Ub. See Ubiquitination immune system and, 25
Ubiquitination (Ub), 279–300 uracil action of, 18
cellular influence of, 283 UV. See Ultraviolet
E2 binding to, 285, 286
histone attachment to chains of poly, 287
Lys-63 conjugation of, 284, 290 V
mono-, 294–295
N-end rule of Rad6 encoded, 287 V(D)J recombination, 309
PCNA and poly-, 298–299 antigen recognition and binding and,
PCNA covalent modification and, 297–298 307–308
poly-, 284, 287, 289–290, 298–299 B cell maturation and, 310
process of, 283, 284 initiation of, 308
PRR and conjugation of, 291 RSS and, 308
Rad6 encoded, 286, 287 T-cell receptor, 308
structure, 286 TdT and, 308–310
SUMO charged surface and, 292
sumoylation and, 294–295
surface-exposed lysine residues and, 286 X
UDG. See Uracil-DNA glycosylase
Ultraviolet (UV) light Xeroderma pimentatosum (XP), 44, 158
apoptosis induced by, 122 mutations in, 217, 218
cancer induced by, 158 Pol defects and, 206
chromatin distributed damaged induced Pol mutations and variations of,
by, 59–60 217–219
circadian clock and ameliorating harmful Pol , nuclear and, 217
effects of, 91 RAD30 genes and, 207
CPD induced by, 205 SHM in, 320
DNA repair and ameliorating harmful UV mutation spectra in, 221
effects of, 91 XP. See Xeroderma pimentatosum
lesions induced by, 63, 121–122 X-ray repair cross-complementing protein 1
mutagenesis induced by, 168, 176, 237 (XRCC1), 24
NER and mutations of, 45 XRCC1. See X-ray repair cross-
NER and sensitivity to, 43–44, 45, 121 complementing protein 1

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ADVANCES IN PROTEIN CHEMISTRY

DNA Repair and Replication

FREDERIC M. RICHARDS DAVID S. EISENBERG

DNA Repair and Replication

This book is printed on acid-free paper.

Copyright ß 2004, Elsevier Inc. All Rights Reserved.

No part of this publication may be reproduced or transmitted in any form or by any

For all information on all Academic Press publications

PRINTED IN THE UNITED STATES OF AMERICA

Base Excision Repair

J. Christopher Fromme and Gregory L. Verdine

Nucleotide Excision Repair in E. Coli and Man

Aziz Sancar and Joyce T. Reardon

Photolyase and Cryptochrome Blue-Light Photoreceptors

Coordination of Repair, Checkpoint, and Cell Death Responses

Jean Y. J. Wang and Sarah K. Cho

Functions of DNA Polymerases

Katarzyna Bebenek and Thomas A. Kunkel

I. DNA Polymerase Families . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

Cellular Functions of DNA Polymerase and Rev1 Protein

DNA Polymerases and

Alexandra Vaisman, Alan R. Lehmann, and Roger Woodgate

Properties and Functions of Escherichia

Robert P. Fuchs, Shingo Fujii, and JØro^ me Wagner

I. DNA Pol IV, the dinB Gene Product . . . . . . . . . . . . . . . . . . . . . . 230

Mammalian Pol : Regulation of Its Expression and Lesion Substrates

Haruo Ohmori, Eiji Ohashi, and Tomoo Ogi

I. Structures of the Genes and Proteins . . . . . . . . . . . . . . . . . . . . . 265

DNA Postreplication Repair Modulated by Ubiquitination and Sumoylation

Landon Pastushok and Wei Xiao

Somatic Hypermutation: A Mutational Panacea

Brigette Tippin, Phuong Pham, Ronda Bransteitter, and Myron F. Goodman

I. Generation of Antibody Diversity . . . . . . . . . . . . . . . . . . . . . . . . . 307

AUTHOR INDEX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337

Error-free mechanisms for DNA damage tolerance have been variously

By J. CHRISTOPHER FROMME* AND GREGORY L. VERDINE*,À

*Department of Molecular and Cellular Biology, Harvard University,

1 Copyright 2004, Elsevier Inc.

its miraculous biologic properties can cause hydrolytic damage to DNA

on the 30 side of the nick. This ‘‘long-patch’’ repair process is thought to

Fig. 3. Reactions catalyzed by apurinic/apyrimidinic (AP) endonucleases. An AP

II. Types of Damage Repaired by BER

Source of damage Example lesion Responsible DNA glycosylases

Water/alkaline Uracil UDG, MUG, SMUG

predominant target of this reaction is cytosine, which undergoes conversion

III. DNA Glycosylases

Fig. 4. Structures of DNA glycosylase-DNA complexes, representing the ﬁve known

B. Distinct Structural Classes

C. Damage Recognition and Searching

AAG Monofunctional AAG Recognition, abasic analog, and

and G. L. Verdine, unpublished), hOGG1 (Bruner et al., 2000), and MutY

Fig. 5. Comparison of 8-oxoguanine binding to the active sites of two different

conformation, makes contact with the Watson–Crick face of oxoG using a

threonine and glutamate sidechains, and recognizes the oxidation status

Tag) with bound substrate analogs and accompanying biochemistry show

MBD4 protein is of particular interest among these evolutionarily wide-

2. Searching for Damage

The mechanism of catalysis by UDG, including events from the initial

IV. Downstream BER Enzymes

B. Short-Patch versus Long-Patch Repair

transpires, the product is chemically identical to the gap resulting from AP

Fig. 8. Modeled structure of a FEN-1/PCNA/DNA complex. This model was

oxidative lesions via generation of free radicals, tends to generate multiple

Mammalian Pol : Regulation of Its Expression and Lesion Substrates

proteins. PARP-1 interacts with DNA polymerase in GST-pulldown assays